U.S. patent application number 15/758004 was filed with the patent office on 2018-09-06 for copi coatomer beta subunit nucleic acid molecules that confer resistance to coleopteran and hemipteran pests.
This patent application is currently assigned to Dow AgroSciences LLC. The applicant listed for this patent is DOW AGROSCIENCES LLC. Invention is credited to Kanika ARORA, Navin ELANGO, Elane FISHILEVICH, Premchand GANDRA, Chaoxian GENG, Matthew J. HENRY, Huarong LI, Kenneth E. NARVA, Murugesan RANGASAMY, Aaron T. WOOSLEY, Sarah E. WORDEN.
Application Number | 20180251779 15/758004 |
Document ID | / |
Family ID | 55747149 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180251779 |
Kind Code |
A1 |
NARVA; Kenneth E. ; et
al. |
September 6, 2018 |
COPI COATOMER BETA SUBUNIT NUCLEIC ACID MOLECULES THAT CONFER
RESISTANCE TO COLEOPTERAN AND HEMIPTERAN PESTS
Abstract
This disclosure concerns nucleic acid molecules and methods of
use thereof for control of insect pests through RNA
interference-mediated inhibition of target coding and transcribed
non-coding sequences in insect pests, including coleopteran and/or
hemipteran pests. The disclosure also concerns methods for making
transgenic plants that express nucleic acid molecules useful for
the control of insect pests, and the plant cells and plants
obtained thereby.
Inventors: |
NARVA; Kenneth E.;
(Zionsville, IN) ; LI; Huarong; (Zionsville,
IN) ; GENG; Chaoxian; (Zionsville, IN) ;
ELANGO; Navin; (Indianapolis, IN) ; HENRY; Matthew
J.; (Indianapolis, IN) ; RANGASAMY; Murugesan;
(Zionsville, IN) ; WOOSLEY; Aaron T.; (Fisher,
IN) ; ARORA; Kanika; (West New York, NJ) ;
GANDRA; Premchand; (Zionsville, IN) ; WORDEN; Sarah
E.; (Indianapolis, IN) ; FISHILEVICH; Elane;
(Indianapolis, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DOW AGROSCIENCES LLC |
Indianapolis |
IN |
US |
|
|
Assignee: |
Dow AgroSciences LLC
Indianapolis
IN
|
Family ID: |
55747149 |
Appl. No.: |
15/758004 |
Filed: |
October 7, 2015 |
PCT Filed: |
October 7, 2015 |
PCT NO: |
PCT/US2015/054478 |
371 Date: |
March 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62063203 |
Oct 13, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/10 20200101;
Y02A 50/30 20180101; C12N 15/113 20130101; A01N 65/20 20130101;
C12N 15/8218 20130101; C12N 15/8286 20130101; Y02A 40/162 20180101;
A01N 57/16 20130101; A01N 65/44 20130101; Y02A 40/146 20180101;
Y02A 50/324 20180101; A01N 57/16 20130101; A01N 63/10 20200101;
A01N 65/00 20130101; A01N 65/20 20130101; A01N 65/44 20130101; A01N
63/10 20200101; A01N 65/00 20130101; A01N 65/20 20130101; A01N
65/44 20130101; A01N 57/16 20130101; A01N 63/10 20200101; A01N
65/00 20130101; A01N 65/20 20130101; A01N 65/44 20130101; A01N
63/10 20200101; A01N 65/00 20130101; A01N 65/20 20130101; A01N
65/44 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/113 20060101 C12N015/113; A01N 63/02 20060101
A01N063/02; A01N 65/44 20060101 A01N065/44; A01N 65/20 20060101
A01N065/20; A01N 57/16 20060101 A01N057/16 |
Claims
1. An isolated nucleic acid comprising at least one polynucleotide
operably linked to a heterologous promoter, wherein the
polynucleotide is selected from the group consisting of: SEQ ID
NO:1; the complement of SEQ ID NO:1; a fragment of at least 15
contiguous nucleotides of SEQ ID NO:1; the complement of a fragment
of at least 15 contiguous nucleotides of SEQ ID NO:1; a native
coding sequence of a Diabrotica organism comprising SEQ ID NO:1;
the complement of a native coding sequence of a Diabrotica organism
comprising SEQ ID NO:1; a fragment of at least 15 contiguous
nucleotides of a native coding sequence of a Diabrotica organism
comprising SEQ ID NO:1; the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Diabrotica
organism comprising SEQ ID NO:1; SEQ ID NO:72; the complement of
SEQ ID NO: 72; a fragment of at least 15 contiguous nucleotides of
SEQ ID NO: 72; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO: 72; a native coding sequence
of a Diabrotica organism comprising SEQ ID NO: 72; the complement
of a native coding sequence of a Diabrotica organism comprising SEQ
ID NO: 72; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Diabrotica organism comprising SEQ ID
NO: 72; the complement of a fragment of at least 15 contiguous
nucleotides of a native coding sequence of a Diabrotica organism
comprising SEQ ID NO: 72; SEQ ID NO:83; the complement of SEQ ID
NO: 83; a fragment of at least 15 contiguous nucleotides of SEQ ID
NO: 83; the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO: 83; a native coding sequence of a
Euschistus organism comprising SEQ ID NO: 83; the complement of a
native coding sequence of a Euschistus organism comprising SEQ ID
NO: 83; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Euschistus organism comprising SEQ ID
NO: 83; the complement of a fragment of at least 15 contiguous
nucleotides of a native coding sequence of a Euschistus organism
comprising SEQ ID NO: 83; and SEQ ID NO:84; the complement of SEQ
ID NO:84; a fragment of at least 15 contiguous nucleotides of SEQ
ID NO:84; the complement of a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:84; a native coding sequence of a
Euschistus organism comprising SEQ ID NO:84; the complement of a
native coding sequence of a Euschistus organism comprising SEQ ID
NO:84; a fragment of at least 15 contiguous nucleotides of a native
coding sequence of a Euschistus organism comprising SEQ ID NO:84;
the complement of a fragment of at least 15 contiguous nucleotides
of a native coding sequence of a Euschistus organism comprising SEQ
ID NO:84.
2. The polynucleotide of claim 1, wherein the polynucleotide is
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:8, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,
SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID
NO:86, and the complements of any of the foregoing.
3. A plant transformation vector comprising the polynucleotide of
claim 1.
4. The polynucleotide of claim 1, wherein the organism is selected
from the group consisting of D. v. virgifera LeConte; D. barberi
Smith and Lawrence; D. u. howardi; D. v. zeae; D. balteata LeConte;
D. u. tenella; D. speciosa Germar; D. u. undecimpunctata
Mannerheim; Euschistus heros (Fabr.) (Neotropical Brown Stink Bug),
Nezara viridula (L.) (Southern Green Stink Bug), Piezodorus
guildinii (Westwood) (Red-banded Stink Bug), Halyomorpha halys
(Stal) (Brown Marmorated Stink Bug), Chinavia hilare (Say) (Green
Stink Bug), Euschistus servus (Say) (Brown Stink Bug), Dichelops
melacanthus (Dallas), Dichelops furcatus (F.), Edessa meditabunda
(F.), Thyanta perditor (F.) (Neotropical Red Shouldered Stink Bug),
Chinavia marginatum (Palisot de Beauvois), Horcias nobilellus
(Berg) (Cotton Bug), Taedia stigmosa (Berg), Dysdercus peruvianus
(Guerin-Meneville), Neomegalotomus parvus (Westwood), Leptoglossus
zonatus (Dallas), Niesthrea sidae (F.), Lygus hesperus (Knight)
(Western Tarnished Plant Bug), and Lygus lineolaris (Palisot de
Beauvois).
5. A ribonucleic acid (RNA) molecule transcribed from the
polynucleotide of claim 1.
6. A double-stranded ribonucleic acid molecule produced from the
expression of the polynucleotide of claim 1.
7. The double-stranded ribonucleic acid molecule of claim 6,
wherein contacting the polynucleotide sequence with a coleopteran
or hemipteran pest inhibits the expression of an endogenous
nucleotide sequence specifically complementary to the
polynucleotide.
8. The double-stranded ribonucleic acid molecule of claim 7,
wherein contacting said ribonucleotide molecule with a coleopteran
or hemipteran pest kills or inhibits the growth, and/or feeding of
the pest.
9. The double stranded RNA of claim 6, comprising a first, a second
and a third RNA segment, wherein the first RNA segment comprises
the polynucleotide, wherein the third RNA segment is linked to the
first RNA segment by the second polynucleotide sequence, and
wherein the third RNA segment is substantially the reverse
complement of the first RNA segment, such that the first and the
third RNA segments hybridize when transcribed into a ribonucleic
acid to form the double-stranded RNA.
10. The RNA of claim 5, selected from the group consisting of a
double-stranded ribonucleic acid molecule and a single-stranded
ribonucleic acid molecule of between about 15 and about 30
nucleotides in length.
11. A plant transformation vector comprising the polynucleotide of
claim 1, wherein the heterologous promoter is functional in a plant
cell.
12. A cell transformed with the polynucleotide of claim 1.
13. The cell of claim 12, wherein the cell is a prokaryotic
cell.
14. The cell of claim 12, wherein the cell is a eukaryotic
cell.
15. The cell of claim 14, wherein the cell is a plant cell.
16. A plant transformed with the polynucleotide of claim 1.
17. A seed of the plant of claim 16, wherein the seed comprises the
polynucleotide.
18. A commodity product produced from the plant of claim 16,
wherein the commodity product comprises a detectable amount of the
polynucleotide.
19. The plant of claim 16, wherein the at least one polynucleotide
is expressed in the plant as a double-stranded ribonucleic acid
molecule.
20.-21. (canceled)
22. The plant of claim 16, wherein the at least one polynucleotide
is expressed in the plant as a ribonucleic acid molecule, and the
ribonucleic acid molecule inhibits the expression of an endogenous
polynucleotide that is specifically complementary to the at least
one polynucleotide when a coleopteran or hemipteran pest ingests a
part of the plant.
23.-52. (canceled)
Description
PRIORITY CLAIMS
[0001] This application is a national stage of application filed
under 35 U.S.C. .sctn. 371 of PCT/US15/054478 filed Oct. 7, 2015,
which claims the benefit of the filing date of U.S. Provisional
Patent Application Ser. No. 62/063,203, filed Oct. 13, 2014, for
"COPI COATOMER BETA SUBUNIT NUCLEIC ACID MOLECULES THAT CONFER
RESISTANCE TO COLEOPTERAN AND HEMIPTERAN PESTS".
FIELD OF THE DISCLOSURE
[0002] The present invention relates generally to genetic control
of plant damage caused by insect pests (e.g., coleopteran pests and
hemipteran pests). In particular embodiments, the present invention
relates to identification of target coding and non-coding
polynucleotides, and the use of recombinant DNA technologies for
post-transcriptionally repressing or inhibiting expression of
target coding and non-coding polynucleotides in the cells of an
insect pest to provide a plant protective effect.
BACKGROUND
[0003] The western corn rootworm (WCR), Diabrotica virgifera
virgifera LeConte, is one of the most devastating corn rootworm
species in North America and is a particular concern in
corn-growing areas of the Midwestern United States. The northern
corn rootworm (NCR), Diabrotica barberi Smith and Lawrence, is a
closely-related species that co-inhabits much of the same range as
WCR. There are several other related subspecies of Diabrotica that
are significant pests in the Americas: the Mexican corn rootworm
(MCR), D. virgifera zeae Krysan and Smith; the southern corn
rootworm (SCR), D. undecimpunctata howardi Barber; D. balteata
LeConte; D. undecimpunctata tenella; D. speciosa Germar; and D. u.
undecimpunctata Mannerheim. The United States Department of
Agriculture estimates that corn rootworms cause $1 billion in lost
revenue each year, including $800 million in yield loss and $200
million in treatment costs.
[0004] Both WCR and NCR are deposited in the soil as eggs during
the summer. The insects remain in the egg stage throughout the
winter. The eggs are oblong, white, and less than 0.004 inches in
length. The larvae hatch in late May or early June, with the
precise timing of egg hatching varying from year to year due to
temperature differences and location. The newly hatched larvae are
white worms that are less than 0.125 inches in length. Once
hatched, the larvae begin to feed on corn roots. Corn rootworms go
through three larval instars. After feeding for several weeks, the
larvae molt into the pupal stage. They pupate in the soil, and then
they emerge from the soil as adults in July and August. Adult
rootworms are about 0.25 inches in length.
[0005] Corn rootworm larvae complete development on corn and
several other species of grasses. Larvae reared on yellow foxtail
emerge later and have a smaller head capsule size as adults than
larvae reared on corn (Ellsbury et al. (2005) Environ. Entomol.
34:627-634). WCR adults feed on corn silk, pollen, and kernels on
exposed ear tips. If WCR adults emerge before corn reproductive
tissues are present, they may feed on leaf tissue, thereby slowing
plant growth and occasionally killing the host plant. However, the
adults will quickly shift to preferred silks and pollen when they
become available. NCR adults also feed on reproductive tissues of
the corn plant, but in contrast rarely feed on corn leaves.
[0006] Most of the rootworm damage in corn is caused by larval
feeding. Newly hatched rootworms initially feed on fine corn root
hairs and burrow into root tips. As the larvae grow larger, they
feed on and burrow into primary roots. When corn rootworms are
abundant, larval feeding often results in the pruning of roots all
the way to the base of the corn stalk. Severe root injury
interferes with the roots' ability to transport water and nutrients
into the plant, reduces plant growth, and results in reduced grain
production, thereby often drastically reducing overall yield.
Severe root injury also often results in lodging of corn plants,
which makes harvest more difficult and further decreases yield.
Furthermore, feeding by adults on the corn reproductive tissues can
result in pruning of silks at the ear tip. If this "silk clipping"
is severe enough during pollen shed, pollination may be
disrupted.
[0007] Control of corn rootworms may be attempted by crop rotation,
chemical insecticides, biopesticides (e.g., the spore-forming
gram-positive bacterium, Bacillus thuringiensis (Bt)), transgenic
plants that express Bt toxins, or a combination thereof. Crop
rotation suffers from the significant disadvantage of placing
unwanted restrictions upon the use of farmland. Moreover,
oviposition of some rootworm species may occur in crop fields other
than corn or extended diapauses results in egg hatching over
multiple years, thereby mitigating the effectiveness of crop
rotation practiced with corn and soybean.
[0008] Chemical insecticides are the most heavily relied upon
strategy for achieving corn rootworm control. Chemical insecticide
use, though, is an imperfect corn rootworm control strategy; over
$1 billion may be lost in the United States each year due to corn
rootworm when the costs of the chemical insecticides are added to
the costs of the rootworm damage that may occur despite the use of
the insecticides. High populations of larvae, heavy rains, and
improper application of the insecticide(s) may all result in
inadequate corn rootworm control. Furthermore, the continual use of
insecticides may select for insecticide-resistant rootworm strains,
as well as raise significant environmental concerns due to the
toxicity of many of them to non-target species.
[0009] Stink bugs and other hemipteran insects (heteroptera)
comprise another important agricultural pest complex. Worldwide
over 50 closely related species of stink bugs are known to cause
crop damage. McPherson & McPherson (2000) Stink bugs of
economic importance in America north of Mexico, CRC Press. These
insects are present in a large number of important crops including
maize, soybean, fruit, vegetables, and cereals.
[0010] Stink bugs go through multiple nymph stages before reaching
the adult stage. The time to develop from eggs to adults is about
30-40 days. Both nymphs and adults feed on sap from soft tissues
into which they also inject digestive enzymes causing extra-oral
tissue digestion and necrosis. Digested plant material and
nutrients are then ingested. Depletion of water and nutrients from
the plant vascular system results in plant tissue damage. Damage to
developing grain and seeds is the most significant as yield and
germination are significantly reduced. Multiple generations occur
in warm climates resulting in significant insect pressure. Current
management of stink bugs relies on insecticide treatment on an
individual field basis. Therefore, alternative management
strategies are urgently needed to minimize ongoing crop losses.
[0011] RNA interference (RNAi) is a process utilizing endogenous
cellular pathways, whereby an interfering RNA (iRNA) molecule
(e.g., a double-stranded RNA (dsRNA) molecule) that is specific for
all, or any portion of adequate size, of a target gene sequence
results in the degradation of the mRNA encoded thereby. In recent
years, RNAi has been used to perform gene "knockdown" in a number
of species and experimental systems; for example, Caenorhabditis
elegans, plants, insect embryos, and cells in tissue culture. See,
e.g., Fire et al. (1998) Nature 391:806-811; Martinez et al. (2002)
Cell 110:563-574; McManus and Sharp (2002) Nature Rev. Genetics
3:737-747.
[0012] RNAi accomplishes degradation of mRNA through an endogenous
pathway including the DICER protein complex. DICER cleaves long
dsRNA molecules into short fragments of approximately 20
nucleotides, termed small interfering RNA (siRNA). The siRNA is
unwound into two single-stranded RNAs: the passenger strand and the
guide strand. The passenger strand is degraded, and the guide
strand is incorporated into the RNA-induced silencing complex
(RISC).
[0013] U.S. Pat. No. 7,612,194 and U.S. Patent Publication Nos.
2007/0050860, 2010/0192265, and 2011/0154545 disclose a library of
9112 expressed sequence tag (EST) sequences isolated from D. v.
virgifera LeConte pupae. It is suggested in U.S. Pat. No. 7,612,194
and U.S. Patent Publication No. 2007/0050860 to operably link to a
promoter a nucleic acid molecule that is complementary to one of
several particular partial sequences of D. v. virgifera
vacuolar-type H.sup.+-ATPase (V-ATPase) disclosed therein for the
expression of anti-sense RNA in plant cells. U.S. Patent
Publication No. 2010/0192265 suggests operably linking a promoter
to a nucleic acid molecule that is complementary to a particular
partial sequence of a D. v. virgifera gene of unknown and
undisclosed function (the partial sequence is stated to be 58%
identical to C56C10.3 gene product in C. elegans) for the
expression of anti-sense RNA in plant cells. U.S. Patent
Publication No. 2011/0154545 suggests operably linking a promoter
to a nucleic acid molecule that is complementary to two particular
partial sequences of D. v. virgifera coatomer beta subunit genes
for the expression of anti-sense RNA in plant cells. Further, U.S.
Pat. No. 7,943,819 discloses a library of 906 expressed sequence
tag (EST) sequences isolated from D. v. virgifera LeConte larvae,
pupae, and dissected midguts, and suggests operably linking a
promoter to a nucleic acid molecule that is complementary to a
particular partial sequence of a D. v. virgifera charged
multivesicular body protein 4b gene for the expression of
double-stranded RNA in plant cells.
[0014] No further suggestion is provided in U.S. Pat. No.
7,612,194, and U.S. Patent Publication Nos. 2007/0050860,
2010/0192265, and 2011/0154545 to use any particular sequence of
the more than nine thousand sequences listed therein for RNA
interference, other than the several particular partial sequences
of V-ATPase and the particular partial sequences of genes of
unknown function. Furthermore, none of U.S. Pat. No. 7,612,194, and
U.S. Patent Publication Nos. 2007/0050860 and 2010/0192265, and
2011/0154545 provides any guidance as to which other of the over
nine thousand sequences provided would be lethal, or even otherwise
useful, in species of corn rootworm when used as dsRNA or siRNA.
U.S. Pat. No. 7,943,819 provides no suggestion to use any
particular sequence of the more than nine hundred sequences listed
therein for RNA interference, other than the particular partial
sequence of a charged multivesicular body protein 4b gene.
Furthermore, U.S. Pat. No. 7,943,819 provides no guidance as to
which other of the over nine hundred sequences provided would be
lethal, or even otherwise useful, in species of corn rootworm when
used as dsRNA or siRNA. U.S. Patent Application Publication No.
U.S. 2013/040173 and PCT Application Publication No. WO 2013/169923
describe the use of a sequence derived from a Diabrotica virgifera
Snf7 gene for RNA interference in maize. (Also disclosed in
Bolognesi et al. (2012) PLOS ONE 7(10): e47534.
doi:10.1371/journal.pone.0047534).
[0015] The overwhelming majority of sequences complementary to corn
rootworm DNAs (such as the foregoing) do not provide a plant
protective effect from species of corn rootworm when used as dsRNA
or siRNA. For example, Baum et al. (2007) Nature Biotechnology
25:1322-1326, describes the effects of inhibiting several WCR gene
targets by RNAi. These authors reported that 8 of the 26 target
genes they tested were not able to provide experimentally
significant coleopteran pest mortality at a very high iRNA (e.g.,
dsRNA) concentration of more than 520 ng/cm.sup.2.
[0016] The authors of U.S. Pat. No. 7,612,194 and U.S. Patent
Publication No. 2007/0050860 made the first report of in planta
RNAi in corn plants targeting the western corn rootworm. Baum et
al. (2007) Nat. Biotechnol. 25(11):1322-6. These authors describe a
high-throughput in vivo dietary RNAi system to screen potential
target genes for developing transgenic RNAi maize. Of an initial
gene pool of 290 targets, only 14 exhibited larval control
potential. One of the most effective double-stranded RNAs (dsRNA)
targeted a gene encoding vacuolar ATPase subunit A (V-ATPase),
resulting in a rapid suppression of corresponding endogenous mRNA
and triggering a specific RNAi response with low concentrations of
dsRNA. Thus, these authors documented for the first time the
potential for in planta RNAi as a possible pest management tool,
while simultaneously demonstrating that effective targets could not
be accurately identified a priori, even from a relatively small set
of candidate genes.
SUMMARY OF THE DISCLOSURE
[0017] Disclosed herein are nucleic acid molecules (e.g., target
genes, DNAs, dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs), and
methods of use thereof, for the control of insect pests, including,
for example, coleopteran pests, such as D. v. virgifera LeConte
(western corn rootworm, "WCR"); D. barberi Smith and Lawrence
(northern corn rootworm, "NCR"); D. u. howardi Barber (southern
corn rootworm, "SCR"); D. v. zeae Krysan and Smith (Mexican corn
rootworm, "MCR"); D. balteata LeConte; D. u. tenella; D. speciosa
Germar; D. u. undecimpunctata Mannerheim, and hemipteran pests,
such as Euschistus heros (Fabr.) (Neotropical Brown Stink Bug,
"BSB"); E. servus (Say) (Brown Stink Bug); Nezara viridula (L.)
(Southern Green Stink Bug); Piezodorus guildinii (Westwood)
(Red-banded Stink Bug); Halyomorpha halys (stat) (Brown Marmorated
Stink Bug); Chinavia hilare (Say) (Green Stink Bug); C. marginatum
(Palisot de Beauvois); Dichelops melacanthus (Dallas); D. furcatus
(F.); Edessa meditabunda (F.); Thyanta perditor (F.) (Neotropical
Red Shouldered Stink Bug); Horcias nobilellus (Berg) (Cotton Bug);
Taedia stigmosa (Berg); Dysdercus peruvianus (Guerin-Meneville);
Neomegalotomus parvus (Westwood); Leptoglossus zonatus (Dallas);
Niesthrea sidae (F.); Lygus hesperus (Knight) (Western Tarnished
Plant Bug); and L. lineolaris (Palisot de Beauvois). In particular
examples, exemplary nucleic acid molecules are disclosed that may
be homologous to at least a portion of one or more native nucleic
acids in an insect pest.
[0018] In these and further examples, the native nucleic acid may
be a target gene, the product of which may be, for example and
without limitation: involved in a metabolic process or involved in
larval/nymph development. In some examples, post-translational
inhibition of the expression of a target gene by a nucleic acid
molecule comprising a polynucleotide homologous thereto may be
lethal in coleopteran and/or hemipteran pests, or result in reduced
growth and/or development thereof. In specific examples, a gene
consisting of the coat protein complex beta subunit (referred to
herein as COPI beta subunit and COPI beta) may be selected as a
target gene for post-transcriptional silencing. In particular
examples, a target gene useful for post-transcriptional inhibition
is the novel gene referred to herein as COPI beta. An isolated
nucleic acid molecule comprising a nucleotide sequence of COPI beta
(SEQ ID NO:1, SEQ ID NO: 72, SEQ ID NO:83, and SEQ ID NO:84); the
complement of COPI beta (SEQ ID NO:1, SEQ ID NO: 72, SEQ ID NO:83,
and SEQ ID NO:84); and fragments of any of the foregoing is
therefore disclosed herein.
[0019] Also disclosed are nucleic acid molecules comprising a
polynucleotide that encodes a polypeptide that is at least about
85% identical to an amino acid sequence within a target gene
product (for example, the product of a gene referred to as COPI
BETA). For example, a nucleic acid molecule may comprise a
polynucleotide encoding a polypeptide that is at least 85%
identical to SEQ ID NO:2, SEQ ID NO:94, or SEQ ID NO:95 (COPI BETA
protein). In particular examples, a nucleic acid molecule comprises
a nucleotide sequence encoding a polypeptide that is at least 85%
identical to an amino acid sequence within a product of COPI BETA.
Further disclosed are nucleic acid molecules comprising a
polynucleotide that is the reverse complement of a polynucleotide
that encodes a polypeptide at least 85% identical to an amino acid
sequence within a target gene product.
[0020] Also disclosed are cDNA polynucleotides that may be used for
the production of iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and
hpRNA) molecules that are complementary to all or part of a
coleopteran and/or hemipteran pest target gene, for example: COPI
beta. In particular embodiments, dsRNAs, siRNAs, miRNAs, shRNAs,
and/or hpRNAs may be produced in vitro, or in vivo by a
genetically-modified organism, such as a plant or bacterium. In
particular examples, cDNA molecules are disclosed that may be used
to produce iRNA molecules that are complementary to all or part of
COPI beta (SEQ ID NO:1, SEQ ID NO: 72, SEQ ID NO:83, and SEQ ID
NO:84).
[0021] Further disclosed are means for inhibiting expression of an
essential gene in a coleopteran and/or hemipteran pest, and means
for providing coleopteran and/or hemipteran pest resistance to a
plant. A means for inhibiting expression of an essential gene in a
coleopteran and/or hemipteran pest is a single- or double-stranded
RNA molecule consisting of at least one of SEQ ID NO:3 (Diabrotica
COPI beta region 1, herein sometimes referred to as COPI beta reg1)
or SEQ ID NO:68 (Diabrotica COPI beta region 2, herein sometimes
referred to as COPI beta reg2), SEQ ID NO:69 (Diabrotica COPI beta
region 3, herein sometimes referred to as COPI beta reg3), SEQ ID
NO:70 (Diabrotica COPI beta version 1, herein sometimes referred to
as COPI beta ver1), SEQ ID NO:71 (Diabrotica COPI beta version 2,
herein sometimes referred to as COPI beta ver2), SEQ ID NO:72
(Diabrotica COPI beta variant 1, herein sometimes referred to as
COPI beta var1), SEQ ID NO:85 (Euschistus heros COPI beta version
1, herein sometimes referred to as BSB_COPI beta-1), SEQ ID NO:86
(Euschistus heros COPI beta version 2, herein sometimes referred to
as BSB_COPI beta-2) or the complement thereof. Functional
equivalents of means for inhibiting expression of an essential gene
in a coleopteran and/or hemipteran pest include single- or
double-stranded RNA molecules that are substantially homologous to
all or part of a WCR or BSB gene comprising SEQ ID NO:1, SEQ ID NO:
72, SEQ ID NO:83, or SEQ ID NO:84. A means for providing
coleopteran and/or hemipteran pest resistance to a plant is a DNA
molecule comprising a nucleic acid sequence encoding a means for
inhibiting expression of an essential gene in a coleopteran and/or
hemipteran pest operably linked to a promoter, wherein the DNA
molecule is capable of being integrated into the genome of a maize
or soybean plant.
[0022] Disclosed are methods for controlling a population of an
insect pest (e.g., a coleopteran or hemipteran pest), comprising
providing to an insect pest (e.g., a coleopteran or hemipteran
pest) an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA)
molecule that functions upon being taken up by the pest to inhibit
a biological function within the pest, wherein the iRNA molecule
comprises all or part of a nucleotide sequence selected from the
group consisting of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ
ID NO:84, SEQ ID NO:85, and SEQ ID NO:86; the complement of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID
NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and
SEQ ID NO:86; a native coding sequence of a Diabrotica organism
(e.g., WCR) or hemipteran organism (e.g. BSB) comprising all or
part of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ
ID NO:84, SEQ ID NO:85, and SEQ ID NO:86; the complement of a
native coding sequence of a Diabrotica or hemipteran organism
comprising all or part of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ
ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86; a native
non-coding sequence of a Diabrotica or hemipteran organism that is
transcribed into a native RNA molecule comprising all or part of
any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:68, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:84, SEQ
ID NO:85, and SEQ ID NO:86; and the complement of a native
non-coding sequence of a Diabrotica organism or hemipteran organism
that is transcribed into a native RNA molecule comprising all or
part of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ
ID NO:84, SEQ ID NO:85, and SEQ ID NO:86.
[0023] Also disclosed herein are methods wherein dsRNAs, siRNAs,
miRNAs, shRNAs and/or hpRNAs may be provided to a coleopteran
and/or hemipteran pest in a diet-based assay, or in
genetically-modified plant cells expressing the dsRNAs, siRNAs,
miRNAs, shRNAs and/or hpRNAs. In these and further examples, the
dsRNAs, siRNAs, miRNAs, shRNAs and/or hpRNAs may be ingested by
coleopteran larvae and/or hemipteran pest nymph. Ingestion of
dsRNAs, siRNA, miRNAs, shRNAs and/or hpRNAs of the invention may
then result in RNAi in the larvae/nymph, which in turn may result
in silencing of a gene essential for viability of the coleopteran
and/or hemipteran pest and leading ultimately to larval/nymph
mortality. Thus, methods are disclosed wherein nucleic acid
molecules comprising exemplary nucleic acid sequence(s) useful for
control of coleopteran and/or hemipteran pests are provided to a
coleopteran and/or hemipteran pest. In particular examples, the
coleopteran and/or hemipteran pest controlled by use of nucleic
acid molecules of the invention may be WCR, NCR, SCR, MCR, D.
balteata, D. u. tenella, D. speciosa, D. u. undecimpunctata,
Euschistus heros, E. servus, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Chinavia hilare, C. marginatum, Dichelops
melacanthus, D. furcatus, Edessa meditabunda, Thyanta perditor,
Horcias nobilellus, Taedia stigmosa, Dysdercus peruvianus,
Neomegalotomus parvus, Leptoglossus zonatus, Niesthrea sidae,
and/or Lygus lineolaris.
[0024] The foregoing and other features will become more apparent
from the following Detailed Description of several embodiments,
which proceeds with reference to the accompanying Figures.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 includes a depiction of the strategy used to generate
dsRNA from a single transcription template with a single pair of
primers.
[0026] FIG. 2 includes a depiction of the strategy used to generate
dsRNA from two transcription templates.
SEQUENCE LISTING
[0027] The nucleic acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, as defined in 37 C.F.R. .sctn. 1.822. The nucleic
acid and amino acid sequences listed define molecules (i.e.,
polynucleotides and polypeptides, respectively) having the
nucleotide and amino acid monomers arranged in the manner
described. The nucleic acid and amino acid sequences listed also
each define a genus of polynucleotides or polypeptides that
comprise the nucleotide and amino acid monomers arranged in the
manner described. In view of the redundancy of the genetic code, it
will be understood that a nucleotide sequence including a coding
sequence also describes the genus of polynucleotides encoding the
same polypeptide as a polynucleotide consisting of the reference
sequence. It will further be understood that an amino acid sequence
describes the genus of polynucleotide ORFs encoding that
polypeptide.
[0028] Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand. As the complement and reverse complement
of a primary nucleic acid sequence are necessarily disclosed by the
primary sequence, the complementary sequence and reverse
complementary sequence of a nucleic acid sequence are included by
any reference to the nucleic acid sequence, unless it is explicitly
stated to be otherwise (or it is clear to be otherwise from the
context in which the sequence appears). Furthermore, as it is
understood in the art that the nucleotide sequence of an RNA strand
is determined by the sequence of the DNA from which it was
transcribed (but for the substitution of uracil (U) nucleobases for
thymine (T)), an RNA sequence is included by any reference to the
DNA sequence encoding it. In the accompanying sequence listing:
[0029] SEQ ID NO:1 shows a DNA sequence comprising COPI beta
subunit from Diabrotica virgifera.
[0030] SEQ ID NO:2 shows an amino acid sequence of a COPI beta
protein from Diabrotica virgifera.
[0031] SEQ ID NO:3 shows a DNA sequence of COPI beta reg1 (region
1) from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0032] SEQ ID NO:4 shows a DNA sequence of a T7 phage promoter.
[0033] SEQ ID NO:5 shows a DNA sequence of a YFP coding region
segment that was used for in vitro dsRNA synthesis (T7 promoter
sequences at 5' and 3' ends not shown).
[0034] SEQ ID NOs:6 and 7 show primers used to amplify portions of
a COPI beta subunit sequence from Diabrotica virgifera comprising
COPI beta reg1 and COPI beta reg2.
[0035] SEQ ID NO:8 presents a COPI beta hairpin v2-RNA-forming
sequence from Diabrotica virgifera as found in pDAB117219. Upper
case bases are COPI beta sense strand, underlined lower case bases
comprise an ST-LS1 intron, non-underlined lower case bases are COPI
beta antisense strand.
TABLE-US-00001 ATTTTGGGAGAATACGCGAGTACTGCTTCCGATATAGAAGTTATCGTTGG
AGAAATTAACAGATTGTTGGGTGAAGGATCCCTCGTTGAAGCTGAGCAGA
gactagtaccggttgggaaaggtatgtttctgcttctacctttgatatat
atataataattatcactaattagtagtaatatagtatttcaagtattttt
ttcaaaataaaagaatgtagtatatagctattgcttttctgtagtttata
agtgtgtatattttaatttataacttttctaatatatgaccaaaacatgg
tgatgtgcaggttgatccgcggttatctgctcagcttcaacgagggatcc
ttcacccaacaatctgttaatttctccaacgataacttctatatcggaag
cagtactcgcgtattctcccaaaat
[0036] SEQ ID NO:9 shows a YFP hairpin-RNA-forming sequence v2 as
found in pDAB110853. Upper case bases are YFP sense strand,
underlined bases comprise an ST-LS1 intron, lower case,
non-underlined bases are YFP antisense strand.
TABLE-US-00002 ATGTCATCTGGAGCACTTCTCTTTCATGGGAAGATTCCTTACGTTGTGGA
GATGGAAGGGAATGTTGATGGCCACACCTTTAGCATACGTGGGAAAGGCT
ACGGAGATGCCTCAGTGGGAAAGgactagtaccggttgggaaaggtatgt
ttctgcttctacctttgatatatatataataattatcactaattagtagt
aatatagtatttcaagtatttttttcaaaataaaagaatgtagtatatag
ctattgcttttctgtagtttataagtgtgtatattttaatttataacttt
tctaatatatgaccaaaacatggtgatgtgcaggttgatccgcggttact
ttcccactgaggcatctccgtagcctttcccacgtatgctaaaggtgtgg
ccatcaacattcccttccatctccacaacgtaaggaatcttcccatgaaa
gagaagtgctccagatgacat
[0037] SEQ ID NO:10 shows a DNA sequence comprising an ST-LS1
intron.
[0038] SEQ ID NO:11 shows a YFP protein coding sequence as found in
pDAB101556.
[0039] SEQ ID NO:12 shows a DNA sequence of Annexin region 1.
[0040] SEQ ID NO:13 shows a DNA sequence of Annexin region 2.
[0041] SEQ ID NO:14 shows a DNA sequence of Beta spectrin 2 region
1.
[0042] SEQ ID NO:15 shows a DNA sequence of Beta spectrin 2 region
2.
[0043] SEQ ID NO:16 shows a DNA sequence of mtRP-L4 region 1.
[0044] SEQ ID NO:17 shows a DNA sequence of mtRP-L4 region 2.
[0045] SEQ ID NOs:18 to 45 show primers used to amplify gene
regions of YFP, Annexin, Beta spectrin 2, and mtRP-L4 for dsRNA
synthesis.
[0046] SEQ ID NO:46 shows a maize DNA sequence encoding a
TIP41-like protein.
[0047] SEQ ID NO:47 shows a DNA sequence of oligonucleotide
T20NV.
[0048] SEQ ID NOs:48 to 52 show sequences of primers and probes
used to measure maize transcript levels.
[0049] SEQ ID NO:53 shows a DNA sequence of a portion of a SpecR
coding region used for binary vector backbone detection.
[0050] SEQ ID NO:54 shows a DNA sequence of a portion of an AAD1
coding region used for genomic copy number analysis.
[0051] SEQ ID NO:55 shows a DNA sequence of a maize invertase
gene.
[0052] SEQ ID NOs:56 to 64 show sequences of primers and probes
used for gene copy number analyses.
[0053] SEQ ID NOs:65 to 67 show sequences of primers and probes
used for maize expression analysis.
[0054] SEQ ID NO:68 shows a DNA sequence of COPI beta reg2 (region
2) from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0055] SEQ ID NO:69 shows a DNA sequence of COPI beta reg3 (region
3) from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0056] SEQ ID NO:70 shows a DNA sequence of COPI beta ver1 (version
1) from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0057] SEQ ID NO:71 shows a DNA sequence of COPI beta ver2 (version
2) from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0058] SEQ ID NO:72 shows a DNA sequence of COPI beta var1 (variant
1) from Diabrotica virgifera that was used for in vitro dsRNA
synthesis (T7 promoter sequences at 5' and 3' ends not shown).
[0059] SEQ ID NO:73 to 82 show primers used to amplify portions of
a COPI beta sequence from Diabrotica virgifera comprising COPI beta
reg2, COPI beta reg3, COPI beta ver1, COPI beta ver2, COPI beta
var1.
[0060] SEQ ID NO:83 shows an exemplary DNA sequence of BSB COPI
beta transcript 1 from a Neotropical Brown Stink Bug (Euschistus
heros). This transcript was used to generate BSB_COPI beta-1
dsRNA.
[0061] SEQ ID NO:84 shows an exemplary DNA sequence of BSB COPI
beta transcript 2 from a Neotropical Brown Stink Bug (Euschistus
heros). This transcript was used to generate BSB_COPI beta-2
dsRNA.
[0062] SEQ ID NO:85 shows a DNA sequence of BSB_COPI beta-1 from
Euschistus heros that was used for in vitro dsRNA synthesis (T7
promoter sequences at 5' and 3' ends not shown).
[0063] SEQ ID NO:86 shows a DNA sequence of BSB_COPI beta-2 from
Euschistus heros that was used for in vitro dsRNA synthesis (T7
promoter sequences at 5' and 3' ends not shown).
[0064] SEQ ID NO:87 to 90 show primers used to amplify portions of
a Euschistus heros COPI beta sequence comprising BSB_COPI beta-1
and BSB_COPI beta-2.
[0065] SEQ ID NO:91 is the sense strand of YFP-targeted dsRNA:
YFPv2
[0066] SEQ ID NO:92 to 93 show primers used to amplify portions of
a YFP-targeted dsRNA: YFPv2
[0067] SEQ ID NO:94 shows an amino acid sequence of a Euschistus
heros COPI beta transcript 1 protein.
[0068] SEQ ID NO:95 shows an amino acid sequence of a Euschistus
heros COPI beta transcript 2 protein.
[0069] SEQ ID NO:96 presents YFP hairpin sequence (YFP v2-1). Upper
case bases are YFP sense strand, underlined lower case bases
comprise an RTM1 intron, non-underlined lower case bases are YFP
antisense strand.
TABLE-US-00003 ATGTCATCTGGAGCACTTCTCTTTCATGGGAAGATTCCTTACGTTGTGGA
GATGGAAGGGAATGTTGATGGCCACACCTTTAGCATACGTGGGAAAGGCT
ACGGAGATGCCTCAGTGGGAAAGtccggcaacatgtttgacgtttgtttg
acgttgtaagtctgatttttgactcttcttttttctccgtcacaatttct
acttccaactaaaatgctaagaacatggttataactttttttttataact
taatatgtgatttggacccagcagatagagctcattactttcccactgag
gcatctccgtagcctttcccacgtatgctaaaggtgtggccatcaacatt
cccttccatctccacaacgtaaggaatcttcccatgaaagagaagtgctc cagatgacat
DETAILED DESCRIPTION
I. Overview of Several Embodiments
[0070] Disclosed herein are methods and compositions for genetic
control of insect (e.g., coleopteran and/or hemipteran) pest
infestations. Methods for identifying one or more gene(s) essential
to the lifecycle of an insect pest for use as a target gene for
RNAi-mediated control of an insect pest population are also
provided. DNA plasmid vectors encoding an RNA molecule may be
designed to suppress one or more target gene(s) essential for
growth, survival, and/or development. In some embodiments, the RNA
molecule may be capable of forming dsRNA molecules. In some
embodiments, methods are provided for post-transcriptional
repression of expression or inhibition of a target gene via nucleic
acid molecules that are complementary to a coding or non-coding
sequence of the target gene in an insect pest. In these and further
embodiments, a pest may ingest one or more dsRNA, siRNA, shRNA,
miRNA, and/or hpRNA molecules transcribed from all or a portion of
a nucleic acid molecule that is complementary to a coding or
non-coding sequence of a target gene, thereby providing a
plant-protective effect.
[0071] Thus, some embodiments involve sequence-specific inhibition
of expression of target gene products, using iRNA (e.g., dsRNA,
siRNA, shRNA, miRNA and/or hpRNA) that is complementary to coding
and/or non-coding sequences of the target gene(s) to achieve at
least partial control of an insect (e.g., coleopteran and/or
hemipteran) pest. Disclosed is a set of isolated and purified
nucleic acid molecules comprising a polynucleotide, for example, as
set forth in any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ
ID NO:84, SEQ ID NO:85, SEQ ID NO:86, and fragments thereof. In
some embodiments, a stabilized dsRNA molecule may be expressed from
this sequence, fragments thereof, or a gene comprising one of these
sequences, for the post-transcriptional silencing or inhibition of
a target gene. In certain embodiments, isolated and purified
nucleic acid molecules comprise all or part of SEQ ID NO: 1. In
other embodiments, isolated and purified nucleic acid molecules
comprise all or part of SEQ ID NO:3. In still further embodiments,
isolated and purified nucleic acid molecules comprise all or part
of SEQ ID NO:68. In other embodiments, isolated and purified
nucleic acid molecules comprise all or part of SEQ ID NO:69. In yet
other embodiments, isolated and purified nucleic acid molecules
comprise all or part of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,
SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86.
[0072] Some embodiments involve a recombinant host cell (e.g., a
plant cell) having in its genome at least one recombinant DNA
encoding at least one iRNA (e.g., dsRNA) molecule(s). In particular
embodiments, the dsRNA molecule(s) may be produced when ingested by
a coleopteran and/or hemipteran pest to post-transcriptionally
silence or inhibit the expression of a target gene in the pest. The
recombinant DNA may comprise, for example, any of SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,
SEQ ID NO:72 SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID
NO:86; fragments of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:68,
SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID NO:86; or a partial
sequence of a gene comprising one or more of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ
ID NO:72, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, or SEQ ID
NO:86; or complements thereof.
[0073] Some embodiments involve a recombinant host cell having in
its genome a recombinant DNA encoding at least one iRNA (e.g.,
dsRNA) molecule(s) comprising all or part of an RNA encoded by SEQ
ID NO:1, SEQ ID NO:72, SEQ ID NO:83, and/or SEQ ID NO:84. and/or
the complement thereof When ingested by an insect (e.g.,
coleopteran and/or hemipteran) pest, the iRNA molecule(s) may
silence or inhibit the expression of a target gene comprising SEQ
ID NO:1, SEQ ID NO:72, SEQ ID NO:83, and/or SEQ ID NO:84, in the
coleopteran and/or hemipteran pest, and thereby result in cessation
of growth, development, and/or feeding in the coleopteran and/or
hemipteran pest.
[0074] In some embodiments, a recombinant host cell having in its
genome at least one recombinant DNA encoding at least one RNA
molecule capable of forming a dsRNA molecule may be a transformed
plant cell. Some embodiments involve transgenic plants comprising
such a transformed plant cell. In addition to such transgenic
plants, progeny plants of any transgenic plant generation,
transgenic seeds, and transgenic plant products, are all provided,
each of which comprises recombinant DNA(s). In particular
embodiments, an RNA molecule capable of forming a dsRNA molecule
may be expressed in a transgenic plant cell. Therefore, in these
and other embodiments, a dsRNA molecule may be isolated from a
transgenic plant cell. In particular embodiments, the transgenic
plant is a plant selected from the group comprising corn (Zea
mays), soybean (Glycine max), and plants of the family Poaceae.
[0075] Some embodiments involve a method for modulating the
expression of a target gene in an insect (e.g., coleopteran and/or
hemipteran) pest cell. In these and other embodiments, a nucleic
acid molecule may be provided, wherein the nucleic acid molecule
comprises a polynucleotide encoding an RNA molecule capable of
forming a dsRNA molecule. In particular embodiments, a
polynucleotide encoding an RNA molecule capable of forming a dsRNA
molecule may be operatively linked to a promoter, and may also be
operatively linked to a transcription termination sequence. In
particular embodiments, a method for modulating the expression of a
target gene in an insect pest cell may comprise: (a) transforming a
plant cell with a vector comprising a polynucleotide encoding an
RNA molecule capable of forming a dsRNA molecule; (b) culturing the
transformed plant cell under conditions sufficient to allow for
development of a plant cell culture comprising a plurality of
transformed plant cells; (c) selecting for a transformed plant cell
that has integrated the vector into its genome; and (d) determining
that the selected transformed plant cell comprises the RNA molecule
capable of forming a dsRNA molecule encoded by the polynucleotide
of the vector. A plant may be regenerated from a plant cell that
has the vector integrated in its genome and comprises the dsRNA
molecule encoded by the polynucleotide of the vector.
[0076] Thus, also disclosed is a transgenic plant comprising a
vector having a polynucleotide encoding an RNA molecule capable of
forming a dsRNA molecule integrated in its genome, wherein the
transgenic plant comprises the dsRNA molecule encoded by the
polynucleotide of the vector. In particular embodiments, expression
of an RNA molecule capable of forming a dsRNA molecule in the plant
is sufficient to modulate the expression of a target gene in a cell
of an insect (e.g., coleopteran or hemipteran) pest that contacts
the transformed plant or plant cell (for example, by feeding on the
transformed plant, a part of the plant (e.g., root) or plant cell),
such that growth and/or survival of the pest is inhibited.
Transgenic plants disclosed herein may display resistance and/or
enhanced tolerance to insect pest infestations. Particular
transgenic plants may display resistance and/or enhanced protection
from one or more coleopteran and/or hemipteran pest(s) selected
from the group consisting of: WCR; NCR; SCR; MCR; D. balteata
LeConte; D. u. tenella; D. speciosa Germar; D. u. undecimpunctata
Mannerheim; Euschistus heros; Piezodorus guildinii; Halyomorpha
halys; Nezara viridula; Chinavia hilare; Euschistus servus;
Dichelops melacanthus; Dichelops furcatus; Edessa meditabunda;
Thyanta perditor; Chinavia marginatum; Horcias nobilellus; Taedia
stigmosa; Dysdercus peruvianus; Neomegalotomus parvus; Leptoglossus
zonatus; Niesthrea sidae; Lygus hesperus; and Lygus lineolaris.
[0077] Also disclosed herein are methods for delivery of control
agents, such as an iRNA molecule, to an insect (e.g., coleopteran
and/or hemipteran) pest. Such control agents may cause, directly or
indirectly, an impairment in the ability of an insect pest
population to feed, grow or otherwise cause damage in a host. In
some embodiments, a method is provided comprising delivery of a
stabilized dsRNA molecule to an insect pest to suppress at least
one target gene in the pest, thereby causing RNAi and reducing or
eliminating plant damage in a pest host. In some embodiments, a
method of inhibiting expression of a target gene in the insect pest
may result in cessation of growth, survival, and/or development, in
the pest.
[0078] In some embodiments, compositions (e.g., a topical
composition) are provided that comprise an iRNA (e.g., dsRNA)
molecule for use with plants, animals, and/or the environment of a
plant or animal to achieve the elimination or reduction of an
insect (e.g., coleopteran and/or hemipteran) pest infestation. In
particular embodiments, the composition may be a nutritional
composition or food source to be fed to the insect pest. Some
embodiments comprise making the nutritional composition or food
source available to the pest. Ingestion of a composition comprising
iRNA molecules may result in the uptake of the molecules by one or
more cells of the pest, which may in turn result in the inhibition
of expression of at least one target gene in cell(s) of the pest.
Ingestion of or damage to a plant or plant cell by an insect pest
infestation may be limited or eliminated in or on any host tissue
or environment in which the pest is present by providing one or
more compositions comprising an iRNA molecule in the host of the
pest.
[0079] The compositions and methods disclosed herein may be used
together in combinations with other iRNA molecules directed to
different targets (e.g., RAS Opposite or ROP (U.S. Patent
Application Publication No. 20150176025) and RNAPII (U.S. Patent
Application Publication No. 20150176009). The potential to affect
multiple target sequences in a pest, for example in larvae, may
increase efficacy and also improve sustainable approaches to insect
pest management involving iRNA technologies. The compositions and
methods disclosed herein may also be used together in combinations
with other methods and compositions for controlling damage by
insect (e.g., coleopteran and/or hemipteran) pests. For example, an
iRNA molecule as described herein for protecting plants from insect
pests may be used in a method comprising the additional use of one
or more chemical agents effective against an insect pest,
biopesticides effective against such a pest, crop rotation,
recombinant genetic techniques that exhibit features different from
the features of RNAi-mediated methods and RNAi compositions (e.g.,
recombinant production of proteins in plants that are harmful to an
insect pest (e.g., Bt toxins)).
II. Abbreviations
[0080] BSB Neotropical brown stink bug (Euschistus heros) [0081]
dsRNA double-stranded ribonucleic acid [0082] EST expressed
sequence tag [0083] GI growth inhibition [0084] NCBI National
Center for Biotechnology Information [0085] gDNA genomic DNA [0086]
iRNA inhibitory ribonucleic acid [0087] ORF open reading frame
[0088] RNAi ribonucleic acid interference [0089] miRNA micro
ribonucleic acid [0090] siRNA small inhibitory ribonucleic acid
[0091] shRNA short hairpin ribonucleic acid [0092] hpRNA hairpin
ribonucleic acid [0093] UTR untranslated region [0094] WCR western
corn rootworm (Diabrotica virgifera virgifera LeConte) [0095] NCR
northern corn rootworm (Diabrotica barberi Smith and Lawrence)
[0096] MCR Mexican corn rootworm (Diabrotica virgifera zeae Krysan
and Smith) [0097] PCR Polymerase chain reaction [0098] qPCR
quantative polymerase chain reaction [0099] RISC RNA-induced
Silencing Complex [0100] SCR southern corn rootworm (Diabrotica
undecimpunctata howardi Barber) [0101] SEM standard error of the
mean [0102] YFP yellow fluorescent protein
III. Terms
[0103] In the description and tables which follow, a number of
terms are used. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided:
[0104] Coleopteran pest: As used herein, the term "coleopteran
pest" refers to insects of the order Coleoptera, including pest
insects in the genus Diabrotica, which feed upon agricultural crops
and crop products, including corn and other true grasses. In
particular examples, a coleopteran pest is selected from a list
comprising D. v. virgifera LeConte (WCR); D. barberi Smith and
Lawrence (NCR); D. u. howardi (SCR); D. v. zeae (MCR); D. balteata
LeConte; D. u. tenella; D. speciosa Germar; and D. u.
undecimpunctata Mannerheim.
[0105] Contact (with an organism): As used herein, the term
"contact with" or "uptake by" an organism (e.g., a coleopteran or
hemipteran pest), with regard to a nucleic acid molecule, includes
internalization of the nucleic acid molecule into the organism, for
example and without limitation: ingestion of the molecule by the
organism (e.g., by feeding); contacting the organism with a
composition comprising the nucleic acid molecule; and soaking of
organisms with a solution comprising the nucleic acid molecule.
[0106] Contig: As used herein the term "contig" refers to a DNA
sequence that is reconstructed from a set of overlapping DNA
segments derived from a single genetic source.
[0107] Corn plant: As used herein, the term "corn plant" refers to
a plant of the species, Zea mays (maize).
[0108] Expression: As used herein, "expression" of a coding
polynucleotide (for example, a gene or a transgene) refers to the
process by which the coded information of a nucleic acid
transcriptional unit (including, e.g., gDNA or cDNA) is converted
into an operational, non-operational, or structural part of a cell,
often including the synthesis of a protein. Gene expression can be
influenced by external signals; for example, exposure of a cell,
tissue, or organism to an agent that increases or decreases gene
expression. Expression of a gene can also be regulated anywhere in
the pathway from DNA to RNA to protein. Regulation of gene
expression occurs, for example, through controls acting on
transcription, translation, RNA transport and processing,
degradation of intermediary molecules such as mRNA, or through
activation, inactivation, compartmentalization, or degradation of
specific protein molecules after they have been made, or by
combinations thereof. Gene expression can be measured at the RNA
level or the protein level by any method known in the art,
including, without limitation, northern blot, RT-PCR, western blot,
or in vitro, in situ, or in vivo protein activity assay(s).
[0109] Genetic material: As used herein, the term "genetic
material" includes all genes, and nucleic acid molecules, such as
DNA and RNA.
[0110] Hemipteran pest: As used herein, the term "hemipteran pest"
refers to insects of the order Hemiptera, including, for example
and without limitation, insects in the families Pentatomidae,
Miridae, Pyrrhocoridae, Coreidae, Alydidae, and Rhopalidae, which
feed on a wide range of host plants and have piercing and sucking
mouth parts. In particular examples, a hemipteran pest is selected
from the list comprising, Euschistus heros (Fabr.) (Neotropical
Brown Stink Bug), Nezara viridula (L.) (Southern Green Stink Bug),
Piezodorus guildinii (Westwood) (Red-banded Stink Bug), Halyomorpha
halys (stat) (Brown Marmorated Stink Bug), Chinavia hilare (Say)
(Green Stink Bug), Euschistus servus (Say) (Brown Stink Bug),
Dichelops melacanthus (Dallas), Dichelops furcatus (F.), Edessa
meditabunda (F.), Thyanta perditor (F.) (Neotropical Red Shouldered
Stink Bug), Chinavia marginatum (Palisot de Beauvois), Horcias
nobilellus (Berg) (Cotton Bug), Taedia stigmosa (Berg), Dysdercus
peruvianus (Guerin-Meneville), Neomegalotomus parvus (Westwood),
Leptoglossus zonatus (Dallas), Niesthrea sidae (F.), Lygus hesperus
(Knight) (Western Tarnished Plant Bug), and Lygus lineolaris
(Palisot de Beauvois).
[0111] Inhibition: As used herein, the term "inhibition," when used
to describe an effect on a coding polynucleotide (for example, a
gene), refers to a measurable decrease in the cellular level of
mRNA transcribed from the coding polynucleotide and/or peptide,
polypeptide, or protein product of the coding polynucleotide. In
some examples, expression of a coding polynucleotide may be
inhibited such that expression is approximately eliminated.
"Specific inhibition" refers to the inhibition of a target coding
polynucleotide without consequently affecting expression of other
coding polynucleotides (e.g., genes) in the cell wherein the
specific inhibition is being accomplished.
[0112] Insect: As used herein with regard to pests, the term
"insect pest" specifically includes coleopteran insect pests and
hemipteran insect pests. In some embodiments, the term also
includes some other insect pests.
[0113] Isolated: An "isolated" biological component (such as a
nucleic acid or protein) has been substantially separated, produced
apart from, or purified away from other biological components in
the cell of the organism in which the component naturally occurs
(i.e., other chromosomal and extra-chromosomal DNA and RNA, and
proteins), while effecting a chemical or functional change in the
component (e.g., a nucleic acid may be isolated from a chromosome
by breaking chemical bonds connecting the nucleic acid to the
remaining DNA in the chromosome). Nucleic acid molecules and
proteins that have been "isolated" include nucleic acid molecules
and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant
expression in a host cell, as well as chemically-synthesized
nucleic acid molecules, proteins, and peptides.
[0114] Nucleic acid molecule: As used herein, the term "nucleic
acid molecule" may refer to a polymeric form of nucleotides, which
may include both sense and anti-sense strands of RNA, cDNA, gDNA,
and synthetic forms and mixed polymers of the above. A nucleotide
or nucleobase may refer to a ribonucleotide, deoxyribonucleotide,
or a modified form of either type of nucleotide. A "nucleic acid
molecule" as used herein is synonymous with "nucleic acid" and
"polynucleotide." A nucleic acid molecule is usually at least 10
bases in length, unless otherwise specified. By convention, the
nucleotide sequence of a nucleic acid molecule is read from the 5'
to the 3' end of the molecule. The "complement" of a nucleic acid
molecule refers to a polynucleotide having nucleobases that may
form base pairs with the nucleobases of the nucleic acid molecule
(i.e., A-T/U, and G-C).
[0115] Some embodiments include nucleic acids comprising a template
DNA that is transcribed into an RNA molecule that is the complement
of an mRNA molecule. In these embodiments, the complement of the
nucleic acid transcribed into the mRNA molecule is present in the
5' to 3' orientation, such that RNA polymerase (which transcribes
DNA in the 5' to 3' direction) will transcribe a nucleic acid from
the complement that can hybridize to the mRNA molecule. Unless
explicitly stated otherwise, or it is clear to be otherwise from
the context, the term "complement" therefore refers to a
polynucleotide having nucleobases, from 5' to 3', that may form
base pairs with the nucleobases of a reference nucleic acid.
Similarly, unless it is explicitly stated to be otherwise (or it is
clear to be otherwise from the context), the "reverse complement"
of a nucleic acid refers to the complement in reverse orientation.
The foregoing is demonstrated in the following illustration:
TABLE-US-00004 5' ATGATGATG 3' polynucleotide 5' TACTACTAC 3'
"complement" of the polynucleotide 5' CATCATCAT 3' "reverse
complement" of the polynucleotide
[0116] Some embodiments of the invention include hairpin
RNA-forming iRNA molecules. In these iRNAs, both the complement of
a nucleic acid to be targeted by RNA interference and the reverse
complement may be found in the same molecule, such that the
single-stranded RNA molecule may "fold over" and hybridize to
itself over region comprising the complementary and reverse
complementary polynucleotides, as demonstrated in the following
illustration:
TABLE-US-00005 5' AUGAUGAUG-linker polynucleotide-CAUCAUCAU 3',
which hybridizes to form:
[0117] "Nucleic acid molecules" include all polynucleotides, for
example: single- and double-stranded forms of DNA; single-stranded
forms of RNA; and double-stranded forms of RNA (dsRNA). The term
"nucleotide sequence" or "nucleic acid sequence" refers to both the
sense and antisense strands of a nucleic acid as either individual
single strands or in the duplex. The term "ribonucleic acid" (RNA)
is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA),
siRNA (small interfering RNA), mRNA (messenger RNA), miRNA
(micro-RNA), shRNA (small hairpin RNA), hpRNA (hairpin RNA), tRNA
(transfer RNAs, whether charged or discharged with a corresponding
acylated amino acid), and cRNA (complementary RNA). The term
"deoxyribonucleic acid" (DNA) is inclusive of cDNA, gDNA, and
DNA-RNA hybrids. The terms "polynucleotide," "nucleic acid,"
"segments" thereof, and "fragments" thereof will be understood by
those in the art to include, for example, gDNAs; ribosomal RNAs;
transfer RNAs; RNAs; messenger RNAs; operons; smaller engineered
polynucleotides that encode or may be adapted to encode peptides,
polypeptides, or proteins; and structural and/or functional
elements within a nucleic acid molecule that are delineated by
their corresponding nucleotide sequence.
[0118] Oligonucleotide: An oligonucleotide is a short nucleic acid
polymer. Oligonucleotides may be formed by cleavage of longer
nucleic acid segments, or by polymerizing individual nucleotide
precursors. Automated synthesizers allow the synthesis of
oligonucleotides up to several hundred bases in length. Because
oligonucleotides may bind to a complementary nucleic acid, they may
be used as probes for detecting DNA or RNA. Oligonucleotides
composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a
technique for the amplification of DNA and RNA (reverse transcribed
into a cDNA) sequences. In PCR, the oligonucleotide is typically
referred to as a "primer," which allows a DNA polymerase to extend
the oligonucleotide and replicate the complementary strand.
[0119] A nucleic acid molecule may include either or both naturally
occurring and modified nucleotides linked together by naturally
occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically,
or may contain non-natural or derivatized nucleotide bases, as will
be readily appreciated by those of skill in the art. Such
modifications include, for example, labels, methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications (e.g., uncharged
linkages: for example, methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.; charged linkages: for example,
phosphorothioates, phosphorodithioates, etc.; pendent moieties: for
example, peptides; intercalators: for example, acridine, psoralen,
etc.; chelators; alkylators; and modified linkages: for example,
alpha anomeric nucleic acids, etc.). The term "nucleic acid
molecule" also includes any topological conformation, including
single-stranded, double-stranded, partially duplexed, triplexed,
hairpinned, circular, and padlocked conformations.
[0120] As used herein with respect to DNA, the term "coding
sequence", "structural nucleotide sequence", or "structural nucleic
acid molecule" refers to a nucleotide sequence that is ultimately
translated into a polypeptide, via transcription and mRNA, when
placed under the control of appropriate regulatory sequences. With
respect to RNA, the term "coding sequence" refers to a nucleotide
sequence that is translated into a peptide, polypeptide, or
protein. The boundaries of a coding sequence are determined by a
translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus. Coding sequences include, but are not
limited to: genomic DNA; cDNA; EST; and recombinant nucleotide
sequences.
[0121] Genome: As used herein, the term "genome" refers to
chromosomal DNA found within the nucleus of a cell, and also refers
to organelle DNA found within subcellular components of the cell.
In some embodiments of the invention, a DNA molecule may be
introduced into a plant cell such that the DNA molecule is
integrated into the genome of the plant cell. In these and further
embodiments, the DNA molecule may be either integrated into the
nuclear DNA of the plant cell, or integrated into the DNA of the
chloroplast or mitochondrion of the plant cell. The term "genome"
as it applies to bacteria refers to both the chromosome and
plasmids within the bacterial cell. In some embodiments of the
invention, a DNA molecule may be introduced into a bacterium such
that the DNA molecule is integrated into the genome of the
bacterium. In these and further embodiments, the DNA molecule may
be either chromosomally-integrated or located as or in a stable
plasmid.
[0122] Sequence identity: The term "sequence identity" or
"identity", as used herein in the context of two nucleic acid or
polypeptide sequences, refers to the residues in the two sequences
that are the same when aligned for maximum correspondence over a
specified comparison window.
[0123] As used herein, the term "percentage of sequence identity"
may refer to the value determined by comparing two optimally
aligned sequences (e.g., nucleic acid sequences or polypeptide
sequences) over a comparison window, wherein the portion of the
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleotide or amino acid
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the comparison window, and multiplying the
result by 100 to yield the percentage of sequence identity. A
sequence that is identical at every position in comparison to a
reference sequence is said to be 100% identical to the reference
sequence, and vice-versa.
[0124] Methods for aligning sequences for comparison are well-known
in the art. Various programs and alignment algorithms are described
in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482;
Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and
Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS
5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890;
Huang et al. (1992) Comp. Appl. Biosci. 8:155-165; Pearson et al.
(1994) Methods Mol. Biol. 24:307-331; Tatiana et al. (1999) FEMS
Microbiol. Lett. 174:247-250. A detailed consideration of sequence
alignment methods and homology calculations can be found in, e.g.,
Altschul et al. (1990) J. Mol. Biol. 215:403-410.
[0125] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al.
(1990)) is available from several sources, including the National
Center for Biotechnology Information (Bethesda, Md.), and on the
internet, for use in connection with several sequence analysis
programs. A description of how to determine sequence identity using
this program is available on the internet under the "help" section
for BLAST.TM.. For comparisons of nucleic acid sequences, the
"Blast 2 sequences" function of the BLAST.TM. (Blastn) program may
be employed using the default BLOSUM62 matrix set to default
parameters. Nucleic acid sequences with even greater similarity to
the reference sequences will show increasing percentage identity
when assessed by this method.
[0126] Specifically hybridizable/Specifically complementary: As
used herein, the terms "Specifically hybridizable" and
"Specifically complementary" are terms that indicate a sufficient
degree of complementarity such that stable and specific binding
occurs between the nucleic acid molecule and a target nucleic acid
molecule. Hybridization between two nucleic acid molecules involves
the formation of an anti-parallel alignment between the nucleic
acid sequences of the two nucleic acid molecules. The two molecules
are then able to form hydrogen bonds with corresponding bases on
the opposite strand to form a duplex molecule that, if it is
sufficiently stable, is detectable using methods well known in the
art. A nucleic acid molecule need not be 100% complementary to its
target sequence to be specifically hybridizable. However, the
amount of sequence complementarity that must exist for
hybridization to be specific is a function of the hybridization
conditions used.
[0127] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ and/or Mg.sup.++
concentration) of the hybridization will determine the stringency
of hybridization. The ionic strength of the wash buffer and the
wash temperature also influence stringency. Calculations regarding
hybridization conditions required for attaining particular degrees
of stringency are known to those of ordinary skill in the art, and
are discussed, for example, in Sambrook et al. (ed.) Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9
and 11, and updates; and Hames and Higgins (eds.) Nucleic Acid
Hybridization, IRL Press, Oxford, 1985. Further detailed
instruction and guidance with regard to the hybridization of
nucleic acids may be found, for example, in Tijssen, "Overview of
principles of hybridization and the strategy of nucleic acid probe
assays," in Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2,
Elsevier, N Y, 1993; and Ausubel et al., Eds., Current Protocols in
Molecular Biology, Chapter 2, Greene Publishing and
Wiley-Interscience, N Y, 1995, and updates.
[0128] As used herein, "stringent conditions" encompass conditions
under which hybridization will occur only if there is more than 80%
sequence match between the hybridization molecule and a homologous
sequence within the target nucleic acid molecule. "Stringent
conditions" include further particular levels of stringency. Thus,
as used herein, "moderate stringency" conditions are those under
which molecules with more than 80% sequence match (i.e. having less
than 20% mismatch) will hybridize; conditions of "high stringency"
are those under which sequences with more than 90% match (i.e.
having less than 10% mismatch) will hybridize; and conditions of
"very high stringency" are those under which sequences with more
than 95% match (i.e. having less than 5% mismatch) will
hybridize.
[0129] The following are representative, non-limiting hybridization
conditions.
[0130] High Stringency condition (detects sequences that share at
least 90% sequence identity): Hybridization in 5.times.SSC buffer
at 65.degree. C. for 16 hours; wash twice in 2.times.SSC buffer at
room temperature for 15 minutes each; and wash twice in
0.5.times.SSC buffer at 65.degree. C. for 20 minutes each.
[0131] Moderate Stringency condition (detects sequences that share
at least 80% sequence identity): Hybridization in
5.times.-6.times.SSC buffer at 65-70.degree. C. for 16-20 hours;
wash twice in 2.times.SSC buffer at room temperature for 5-20
minutes each; and wash twice in 1.times.SSC buffer at 55-70.degree.
C. for 30 minutes each.
[0132] Non-stringent control condition (sequences that share at
least 50% sequence identity will hybridize): Hybridization in
6.times.SSC buffer at room temperature to 55.degree. C. for 16-20
hours; wash at least twice in 2.times.-3.times.SSC buffer at room
temperature to 55.degree. C. for 20-30 minutes each.
[0133] As used herein, the term "substantially homologous" or
"substantial homology," with regard to a nucleic acid, refers to a
polynucleotide having contiguous nucleobases that hybridize under
stringent conditions to the reference nucleic acid. For example,
nucleic acids that are substantially homologous to a reference
nucleic acid of any of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83
and/or SEQ ID NO:84 are those nucleic acids that hybridize under
stringent conditions (e.g., the Moderate Stringency conditions set
forth, supra) to the reference nucleic acid of any of SEQ ID NO:1,
SEQ ID NO:72, SEQ ID NO:83 and/or SEQ ID NO:84, respectively.
Substantially homologous polynucleotides may have at least 80%
sequence identity. For example, substantially homologous
polynucleotides may have from about 80% to 100% sequence identity,
such as 79%; 80%; about 81%; about 82%; about 83%; about 84%; about
85%; about 86%; about 87%; about 88%; about 89%; about 90%; about
91%; about 92%; about 93%; about 94% about 95%; about 96%; about
97%; about 98%; about 98.5%; about 99%; about 99.5%; and about
100%. The property of substantial homology is closely related to
specific hybridization. For example, a nucleic acid molecule is
specifically hybridizable when there is a sufficient degree of
complementarity to avoid non-specific binding of the nucleic acid
to non-target polynucleotides under conditions where specific
binding is desired, for example, under stringent hybridization
conditions.
[0134] As used herein, the term "ortholog" refers to a gene in two
or more species that has evolved from a common ancestral nucleic
acid, and may retain the same function in the two or more
species.
[0135] As used herein, two nucleic acid molecules are said to
exhibit "complete complementarity" when every nucleotide of a
polynucleotide read in the 5' to 3' direction is complementary to
every nucleotide of the other polynucleotide when read in the 3' to
5' direction. A polynucleotide that is complementary to a reference
polynucleotide will exhibit a sequence identical to the reverse
complement of the reference polynucleotide. These terms and
descriptions are well defined in the art and are easily understood
by those of ordinary skill in the art.
[0136] Operably linked: A first polynucleotide is operably linked
with a second polynucleotide when the first polynucleotide is in a
functional relationship with the second polynucleotide. When
recombinantly produced, operably linked polynucleotides are
generally contiguous, and, where necessary to join two
protein-coding regions, in the same reading frame (e.g., in a
translationally fused ORF). However, nucleic acids need not be
contiguous to be operably linked.
[0137] The term, "operably linked," when used in reference to a
regulatory genetic element and a coding polynucleotide, means that
the regulatory element affects the expression of the linked coding
polynucleotide. "Regulatory elements," or "control elements," refer
to polynucleotides that influence the timing and level/amount of
transcription, RNA processing or stability, or translation of the
associated coding polynucleotide. Regulatory elements may include
promoters; translation leaders; introns; enhancers; stem-loop
structures; repressor binding polynucleotides; polynucleotides with
a termination sequence; polynucleotides with a polyadenylation
recognition sequence; etc. Particular regulatory elements may be
located upstream and/or downstream of a coding polynucleotide
operably linked thereto. Also, particular regulatory elements
operably linked to a coding polynucleotide may be located on the
associated complementary strand of a double-stranded nucleic acid
molecule.
[0138] Promoter: As used herein, the term "promoter" refers to a
region of DNA that may be upstream from the start of transcription,
and that may be involved in recognition and binding of RNA
polymerase and other proteins to initiate transcription. A promoter
may be operably linked to a coding polynucleotide for expression in
a cell, or a promoter may be operably linked to a polynucleotide
encoding a signal peptide which may be operably linked to a coding
polynucleotide for expression in a cell. A "plant promoter" may be
a promoter capable of initiating transcription in plant cells.
Examples of promoters under developmental control include promoters
that preferentially initiate transcription in certain tissues, such
as leaves, roots, seeds, fibers, xylem vessels, tracheids, or
sclerenchyma. Such promoters are referred to as "tissue-preferred".
Promoters which initiate transcription only in certain tissues are
referred to as "tissue-specific". A "cell type-specific" promoter
primarily drives expression in certain cell types in one or more
organs, for example, vascular cells in roots or leaves. An
"inducible" promoter may be a promoter which may be under
environmental control. Examples of environmental conditions that
may initiate transcription by inducible promoters include anaerobic
conditions and the presence of light. Tissue-specific,
tissue-preferred, cell type specific, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter which may be active under
most environmental conditions or in most tissue or cell types.
[0139] Any inducible promoter can be used in some embodiments of
the invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366.
With an inducible promoter, the rate of transcription increases in
response to an inducing agent. Exemplary inducible promoters
include, but are not limited to: Promoters from the ACEI system
that respond to copper; In2 gene from maize that responds to
benzenesulfonamide herbicide safeners; Tet repressor from Tn10; and
the inducible promoter from a steroid hormone gene, the
transcriptional activity of which may be induced by a
glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad.
Sci. USA 88:0421).
[0140] Exemplary constitutive promoters include, but are not
limited to: Promoters from plant viruses, such as the 35S promoter
from Cauliflower Mosaic Virus (CaMV); promoters from rice actin
genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter;
and the ALS promoter, XbaI/NcoI fragment 5' to the Brassica napus
ALS3 structural gene (or a polynucleotide similar to said XbaI/NcoI
fragment) (International PCT Publication No. WO96/30530).
[0141] Additionally, any tissue-specific or tissue-preferred
promoter may be utilized in some embodiments of the invention.
Plants transformed with a nucleic acid molecule comprising a coding
polynucleotide operably linked to a tissue-specific promoter may
produce the product of the coding polynucleotide exclusively, or
preferentially, in a specific tissue. Exemplary tissue-specific or
tissue-preferred promoters include, but are not limited to: A
seed-preferred promoter, such as that from the phaseolin gene; a
leaf-specific and light-induced promoter such as that from cab or
rubisco; an anther-specific promoter such as that from LAT52; a
pollen-specific promoter such as that from Zm13; and a
microspore-preferred promoter such as that from apg.
[0142] Soybean plant: As used herein, the term "soybean plant"
refers to a plant of the species Glycine sp.; for example, G.
max.
[0143] Transformation: As used herein, the term "transformation" or
"transduction" refers to the transfer of one or more nucleic acid
molecule(s) into a cell. A cell is "transformed" by a nucleic acid
molecule transduced into the cell when the nucleic acid molecule
becomes stably replicated by the cell, either by incorporation of
the nucleic acid molecule into the cellular genome, or by episomal
replication. As used herein, the term "transformation" encompasses
all techniques by which a nucleic acid molecule can be introduced
into such a cell. Examples include, but are not limited to:
transfection with viral vectors; transformation with plasmid
vectors; electroporation (Fromm et al. (1986) Nature 319:791-3);
lipofection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA
84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85);
Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl.
Acad. Sci. USA 80:4803-7); direct DNA uptake; and microprojectile
bombardment (Klein et al. (1987) Nature 327:70).
[0144] Transgene: An exogenous nucleic acid. In some examples, a
transgene may be a DNA that encodes one or both strand(s) of an RNA
capable of forming a dsRNA molecule that comprises a polynucleotide
that is complementary to a nucleic acid molecule found in a
coleopteran and/or hemipteran pest. In further examples, a
transgene may be a gene (e.g., a herbicide-tolerance gene, a gene
encoding an industrially or pharmaceutically useful compound, or a
gene encoding a desirable agricultural trait). In these and other
examples, a transgene may contain regulatory elements operably
linked to a coding polynucleotide of the transgene (e.g., a
promoter).
[0145] Vector: A nucleic acid molecule as introduced into a cell,
for example, to produce a transformed cell. A vector may include
genetic elements that permit it to replicate in the host cell, such
as an origin of replication. Examples of vectors include, but are
not limited to: a plasmid; cosmid; bacteriophage; or virus that
carries exogenous DNA into a cell. A vector may also include one or
more genes, including ones that produce antisense molecules, and/or
selectable marker genes and other genetic elements known in the
art. A vector may transduce, transform, or infect a cell, thereby
causing the cell to express the nucleic acid molecules and/or
proteins encoded by the vector. A vector optionally includes
materials to aid in achieving entry of the nucleic acid molecule
into the cell (e.g., a liposome, protein coating, etc.).
[0146] Yield: A stabilized yield of about 100% or greater relative
to the yield of check varieties in the same growing location
growing at the same time and under the same conditions. In
particular embodiments, "improved yield" or "improving yield" means
a cultivar having a stabilized yield of 105% or greater relative to
the yield of check varieties in the same growing location
containing significant densities of the coleopteran and/or
hemipteran pests that are injurious to that crop growing at the
same time and under the same conditions, which pests are targeted
by the compositions and methods herein.
[0147] Unless specifically indicated or implied, the terms "a,"
"an," and "the" signify "at least one," as used herein.
[0148] Unless otherwise specifically explained, all technical and
scientific terms used herein have the same meaning as commonly
understood by those of ordinary skill in the art to which this
disclosure belongs. Definitions of common terms in molecular
biology can be found in, for example, Lewin's Genes X, Jones &
Bartlett Publishers, 2009 (ISBN 10 0763766321); Krebs et al.
(eds.), The Encyclopedia of Molecular Biology, Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Meyers R. A. (ed.), Molecular
Biology and Biotechnology: A Comprehensive Desk Reference, VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percentages are by
weight and all solvent mixture proportions are by volume unless
otherwise noted. All temperatures are in degrees Celsius.
IV. Nucleic Acid Molecules Comprising an Insect Pest
Polynucleotide
[0149] A. Overview
[0150] Described herein are nucleic acid molecules useful for the
control of insect pests. In some examples, the insect pest is a
coleopteran or hemipteran insect pest. Described nucleic acid
molecules include target polynucleotides (e.g., native genes, and
non-coding polynucleotides), dsRNAs, siRNAs, shRNAs, hpRNAs, and
miRNAs. For example, dsRNA, siRNA, miRNA, shRNA, and/or hpRNA
molecules are described in some embodiments that may be
specifically complementary to all or part of one or more native
nucleic acids in a coleopteran and/or hemipteran pest. In these and
further embodiments, the native nucleic acid(s) may be one or more
target gene(s), the product of which may be, for example and
without limitation: involved in larval/nymph development. Nucleic
acid molecules described herein, when introduced into a cell
comprising at least one native nucleic acid(s) to which the nucleic
acid molecules are specifically complementary, may initiate RNAi in
the cell, and consequently reduce or eliminate expression of the
native nucleic acid(s). In some examples, reduction or elimination
of the expression of a target gene by a nucleic acid molecule
specifically complementary thereto may result in reduction or
cessation of growth, development, and/or feeding of the pest.
[0151] In some embodiments, at least one target gene in an insect
pest may be selected, wherein the target gene comprises a COPI beta
(SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83, or SEQ ID NO:84). In
particular examples, a target gene in a coleopteran or hemipteran
pest is selected, wherein the target gene comprises a novel
nucleotide sequence comprising COPI beta (SEQ ID NO:1, SEQ ID
NO:72, SEQ ID NO:83, or SEQ ID NO:84).
[0152] In some embodiments, a target gene may be a nucleic acid
molecule comprising a polynucleotide that can be translated in
silico to a polypeptide comprising a contiguous amino acid sequence
that is at least about 85% identical (e.g., at least 84%, 85%,
about 90%, about 95%, about 96%, about 97%, about 98%, about 99%,
about 100%, or 100% identical) to the amino acid sequence of a
protein product of COPI beta (SEQ ID NO:1, SEQ ID NO:72, SEQ ID
NO:83, or SEQ ID NO:84). A target gene may be any nucleic acid in
an insect pest, the post-transcriptional inhibition of which has a
deleterious effect on the growth and/or survival of the pest, for
example, to provide a protective benefit against the pest to a
plant. In particular examples, a target gene is a nucleic acid
molecule comprising a polynucleotide that can be reverse translated
in silico to a polypeptide comprising a contiguous amino acid
sequence that is at least about 85% identical, about 90% identical,
about 95% identical, about 96% identical, about 97% identical,
about 98% identical, about 99% identical, about 100% identical, or
100% identical to the amino acid sequence of a protein product of
novel nucleotide sequence SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83,
or SEQ ID NO:84.
[0153] Provided in some embodiments are DNAs, the expression of
which results in an RNA molecule comprising a polynucleotide that
is specifically complementary to all or part of a native RNA
molecule that is encoded by a coding polynucleotide in an insect
(e.g., coleopteran and/or hemipteran) pest. In some embodiments,
after ingestion of the expressed RNA molecule by an insect pest,
down-regulation of the coding polynucleotide in cells of the pest
may be obtained. In particular embodiments, down-regulation of the
coding sequence in cells of the insect pest may result in a
deleterious effect on the growth, development, and/or survival of
the pest.
[0154] In some embodiments, target polynucleotides include
transcribed non-coding RNAs, such as 5'UTRs; 3'UTRs; spliced
leaders; introns; outrons (e.g., 5'UTR RNA subsequently modified in
trans splicing); donatrons (e.g., non-coding RNA required to
provide donor sequences for trans splicing); and other non-coding
transcribed RNA of target insect pest genes. Such polynucleotides
may be derived from both mono-cistronic and poly-cistronic
genes.
[0155] Thus, also described herein in connection with some
embodiments are iRNA molecules (e.g., dsRNAs, siRNAs, miRNAs,
shRNAs, and hpRNAs) that comprise at least one polynucleotide that
is specifically complementary to all or part of a target nucleic
acid in an insect (e.g., coleopteran and/or hemipteran) pest. In
some embodiments an iRNA molecule may comprise polynucleotide(s)
that are complementary to all or part of a plurality of target
nucleic acids; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
target nucleic acids. In particular embodiments, an iRNA molecule
may be produced in vitro, or in vivo by a genetically-modified
organism, such as a plant or bacterium. Also disclosed are cDNAs
that may be used for the production of dsRNA molecules, siRNA
molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules
that are specifically complementary to all or part of a target
nucleic acid in an insect pest. Further described are recombinant
DNA constructs for use in achieving stable transformation of
particular host targets. Transformed host targets may express
effective levels of dsRNA, siRNA, miRNA, shRNA, and/or hpRNA
molecules from the recombinant DNA constructs. Therefore, also
described is a plant transformation vector comprising at least one
polynucleotide operably linked to a heterologous promoter
functional in a plant cell, wherein expression of the
polynucleotide(s) results in an RNA molecule comprising a string of
contiguous nucleobases that is specifically complementary to all or
part of a target nucleic acid in an insect pest.
[0156] In particular examples, nucleic acid molecules useful for
the control of insect (e.g., coleopteran and/or hemipteran) pests
may include: all or part of a native nucleic acid isolated from
Diabrotica or hemipteran organism comprising COPI beta (SEQ ID
NO:1, SEQ ID NO:72, SEQ ID NO:83, or SEQ ID NO:84) DNAs; nucleotide
sequences that when expressed result in an RNA molecule comprising
a polynucleotide that is specifically complementary to all or part
of a native RNA molecule that is encoded by COPI beta (SEQ ID NO:1,
SEQ ID NO:72, SEQ ID NO:83, or SEQ ID NO:84); iRNA molecules (e.g.,
dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) that comprise at least
one polynucleotide that is specifically complementary to all or
part of COPI beta (SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83, or SEQ
ID NO:84); cDNA sequences that may be used for the production of
dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules,
and/or hpRNA molecules that are specifically complementary to all
or part of COPI beta (SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83, or
SEQ ID NO:84); and recombinant DNA constructs for use in achieving
stable transformation of particular host targets, wherein a
transformed host target comprises one or more of the foregoing
nucleic acid molecules.
[0157] B. Nucleic Acid Molecules
[0158] The present invention provides, inter alia, iRNA (e.g.,
dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules that inhibit
target gene expression in a cell, tissue, or organ of an insect
(e.g., coleopteran and/or hemipteran) pest; and DNA molecules
capable of being expressed as an iRNA molecule in a cell or
microorganism to inhibit target gene expression in a cell, tissue,
or organ of an insect pest.
[0159] Some embodiments of the invention provide an isolated
nucleic acid molecule comprising at least one (e.g., one, two,
three, or more) polynucleotide(s) selected from the group
consisting of: any of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83, or
SEQ ID NO:84; the complement of any of SEQ ID NO:1, SEQ ID NO:72,
SEQ ID NO:83, or SEQ ID NO:84; a fragment of at least 15 contiguous
nucleotides of any of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83, or
SEQ ID NO:84; the complement of a fragment of at least 15
contiguous nucleotides of any of SEQ ID NO:1, SEQ ID NO:72, SEQ ID
NO:83, or SEQ ID NO:84; a native coding polynucleotide of a
Diabrotica organism (e.g., WCR) comprising SEQ ID NO:1 or SEQ ID
NO:72; a native coding sequence of a hemipteran organism comprising
SEQ ID NO:83 or SEQ ID NO:84; the complement of a native coding
sequence of a Diabrotica organism comprising SEQ ID NO:1 or SEQ ID
NO:72; the complement of a native coding sequence of a hemipteran
organism comprising SEQ ID NO:83 or SEQ ID NO:84; a native
non-coding sequence of a Diabrotica organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:1 or SEQ ID NO:72;
a native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:83 or
SEQ ID NO:84; the complement of a native non-coding sequence of a
Diabrotica organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:1 or SEQ ID NO:72; the complement of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:83 or SEQ ID NO:84;
a fragment of at least 15 contiguous nucleotides of a native coding
polynucleotide of a Diabrotica organism comprising SEQ ID NO:1 or
SEQ ID NO:72; a fragment of at least 15 contiguous nucleotides of a
native coding polynucleotide of a hemipteran organism comprising
SEQ ID NO:83 or SEQ ID NO:84; the complement of a fragment of at
least 15 contiguous nucleotides of a native coding sequence of a
Diabrotica organism comprising SEQ ID NO:1 or SEQ ID NO:72; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:83 or SEQ ID NO:84; a fragment of at least 15 contiguous
nucleotides of a native non-coding sequence of a Diabrotica
organism that is transcribed into a native RNA molecule comprising
SEQ ID NO:1 or SEQ ID NO:72; a fragment of at least 15 contiguous
nucleotides of a native non-coding sequence of a hemipteran
organism that is transcribed into a native RNA molecule comprising
SEQ ID NO:83 or SEQ ID NO:84; the complement of a fragment of at
least 15 contiguous nucleotides of a native non-coding sequence of
a Diabrotica organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:1 or SEQ ID NO:72; and the complement
of a fragment of at least 15 contiguous nucleotides of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:83 or SEQ ID NO:84.
In particular embodiments, contact with or uptake by a coleopteran
and/or hemipteran pest of the isolated polynucleotide inhibits the
growth, development and/or feeding of the pest.
[0160] In some embodiments, a nucleic acid molecule of the
invention may comprise at least one (e.g., one, two, three, or
more) DNA(s) capable of being expressed as an iRNA molecule in a
cell or microorganism to inhibit target gene expression in a cell,
tissue, or organ of a coleopteran and/or hemipteran pest. Such
DNA(s) may be operably linked to a promoter that functions in a
cell comprising the DNA molecule to initiate or enhance the
transcription of the encoded RNA capable of forming a dsRNA
molecule(s). In one embodiment, the at least one (e.g., one, two,
three, or more) DNA(s) may be derived from a polynucleotide
selected from a group comprising SEQ ID NO:1, SEQ ID NO:72, SEQ ID
NO:83 or SEQ ID NO:84. Derivatives of SEQ ID NO:1, SEQ ID NO:72,
SEQ ID NO:83 or SEQ ID NO:84 include fragments of SEQ ID NO:1, SEQ
ID NO:72, SEQ ID NO:83 or SEQ ID NO:84. In some embodiments, such a
fragment may comprise, for example, at least about 15 contiguous
nucleotides of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID
NO:84, or a complement thereof. Thus, such a fragment may comprise,
for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200 or more contiguous nucleotides of SEQ ID
NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84, or a complement
thereof. In some examples, such a fragment may comprise, for
example, at least 19 contiguous nucleotides of SEQ ID NO:1, SEQ ID
NO:72, SEQ ID NO:83 or SEQ ID NO:84, or a complement thereof. Thus,
a fragment of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID
NO:84 may comprise, for example, 15, 16, 17, 18, 19, 20, 21, about
25, (e.g., 22, 23, 24, 25, 26, 27, 28, and 29), about 30, about 40,
(e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45), about 50,
about 60, about 70, about 80, about 90, about 100, about 110, about
120, about 130, about 140, about 150, about 160, about 170, about
180, about 190, about 200 or more contiguous nucleotides of SEQ ID
NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84, or a complement
thereof.
[0161] Some embodiments comprise introducing partially- or
fully-stabilized dsRNA molecules into a coleopteran and/or
hemipteran pest to inhibit expression of a target gene in a cell,
tissue, or organ of the coleopteran and/or hemipteran pest. When
expressed as an iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA,
and hpRNA) and taken up by a coleopteran and/or hemipteran pest,
polynucleotides comprising one or more fragments of any of SEQ ID
NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84 and the
complements thereof, may cause one or more of death, developmental
arrest, growth inhibition, change in sex ratio, reduction in brood
size, cessation of infection, and/or cessation of feeding by a
coleopteran and/or hemipteran pest. For example, in some
embodiments, a dsRNA molecule comprising a nucleotide sequence
including about 15 to about 300 or about 19 to about 300
nucleotides that are substantially homologous to a coleopteran
and/or hemipteran pest target gene sequence and comprising one or
more fragments of a nucleotide sequence comprising SEQ ID NO:1, SEQ
ID NO:72, SEQ ID NO:83 or SEQ ID NO:84 is provided. Expression of
such a dsRNA molecule may, for example, lead to mortality and/or
growth inhibition in a coleopteran and/or hemipteran pest that
takes up the dsRNA molecule.
[0162] In certain embodiments, dsRNA molecules provided by the
invention comprise polynucleotides complementary to a transcript
from a target gene comprising SEQ ID NO:1, SEQ ID NO:72, SEQ ID
NO:83 or SEQ ID NO:84 and/or nucleotide sequences complementary to
a fragment of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID
NO:84, the inhibition of which target gene in an insect pest
results in the reduction or removal of a polypeptide or
polynucleotide agent that is essential for the pest's growth,
development, or other biological function. A selected
polynucleotide may exhibit from about 80% to about 100% sequence
identity to any of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ
ID NO:84, a contiguous fragment of the nucleotide sequence set
forth in SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84,
or the complement of either of the foregoing. For example, a
selected polynucleotide may exhibit 79%; 80%; about 81%; about 82%;
about 83%; about 84%; about 85%; about 86%; about 87%; about 88%;
about 89%; about 90%; about 91%; about 92%; about 93%; about 94%
about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%;
about 99.5%; or about 100% sequence identity to any of SEQ ID NO:1,
SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84, a contiguous fragment
of the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:72,
SEQ ID NO:83 or SEQ ID NO:84, or the complement of either of the
foregoing.
[0163] In some embodiments, a DNA molecule capable of being
expressed as an iRNA molecule in a cell or microorganism to inhibit
target gene expression may comprise a single polynucleotide that is
specifically complementary to all or part of a native
polynucleotide found in one or more target insect pest species
(e.g., a coleopteran or hemipteran pest species), or the DNA
molecule can be constructed as a chimera from a plurality of such
specifically complementary polynucleotides.
[0164] In some embodiments, a nucleic acid molecule may comprise a
first and a second polynucleotide separated by a "spacer." A spacer
may be a region comprising any sequence of nucleotides that
facilitates secondary structure formation between the first and
second polynucleotides, where this is desired. In one embodiment,
the spacer is part of a sense or antisense coding polynucleotide
for mRNA. The spacer may alternatively comprise any combination of
nucleotides or homologues thereof that are capable of being linked
covalently to a nucleic acid molecule. In some examples, the spacer
may be an intron (e.g., an ST-LS1 intron or a RTM1 intron).
[0165] For example, in some embodiments, the DNA molecule may
comprise a polynucleotide coding for one or more different iRNA
molecules, wherein each of the different iRNA molecules comprises a
first polynucleotide and a second polynucleotide, wherein the first
and second polynucleotides are complementary to each other. The
first and second polynucleotides may be connected within an RNA
molecule by a spacer. The spacer may constitute part of the first
polynucleotide or the second polynucleotide. Expression of an RNA
molecule comprising the first and second nucleotide polynucleotides
may lead to the formation of a dsRNA molecule, by specific
intramolecular base-pairing of the first and second nucleotide
polynucleotides. The first polynucleotide or the second
polynucleotide may be substantially identical to a polynucleotide
(e.g., a target gene, or transcribed non-coding polynucleotide)
native to an insect pest (e.g., a coleopteran or hemipteran pest),
a derivative thereof, or a complementary polynucleotide
thereto.
[0166] dsRNA nucleic acid molecules comprise double strands of
polymerized ribonucleotides, and may include modifications to
either the phosphate-sugar backbone or the nucleoside.
Modifications in RNA structure may be tailored to allow specific
inhibition. In one embodiment, dsRNA molecules may be modified
through a ubiquitous enzymatic process so that siRNA molecules may
be generated. This enzymatic process may utilize an RNase III
enzyme, such as DICER in eukaryotes, either in vitro or in vivo.
See Elbashir et al. (2001) Nature 411:494-8; and Hamilton and
Baulcombe (1999) Science 286(5441):950-2. DICER or
functionally-equivalent RNase III enzymes cleave larger dsRNA
strands and/or hpRNA molecules into smaller oligonucleotides (e.g.,
siRNAs), each of which is about 19-25 nucleotides in length. The
siRNA molecules produced by these enzymes have 2 to 3 nucleotide 3'
overhangs, and 5' phosphate and 3' hydroxyl termini. The siRNA
molecules generated by RNase III enzymes are unwound and separated
into single-stranded RNA in the cell. The siRNA molecules then
specifically hybridize with RNAs transcribed from a target gene,
and both RNA molecules are subsequently degraded by an inherent
cellular RNA-degrading mechanism. This process may result in the
effective degradation or removal of the RNA encoded by the target
gene in the target organism. The outcome is the
post-transcriptional silencing of the targeted gene. In some
embodiments, siRNA molecules produced by endogenous RNase III
enzymes from heterologous nucleic acid molecules may efficiently
mediate the down-regulation of target genes in coleopteran and/or
hemipteran pests.
[0167] In some embodiments, a nucleic acid molecule may include at
least one non-naturally occurring polynucleotide that can be
transcribed into a single-stranded RNA molecule capable of forming
a dsRNA molecule in vivo through intermolecular hybridization. Such
dsRNAs typically self-assemble, and can be provided in the
nutrition source of an insect (e.g., coleopteran or hemipteran)
pest to achieve the post-transcriptional inhibition of a target
gene. In these and further embodiments, a nucleic acid molecule may
comprise two different non-naturally occurring polynucleotides,
each of which is specifically complementary to a different target
gene in an insect pest. When such a nucleic acid molecule is
provided as a dsRNA molecule to, for example, a coleopteran and/or
hemipteran pest, the dsRNA molecule inhibits the expression of at
least two different target genes in the pest.
[0168] C. Obtaining Nucleic Acid Molecules
[0169] A variety of polynucleotides in insect (e.g., coleopteran
and hemipteran) pests may be used as targets for the design of
nucleic acid molecules, such as iRNAs and DNA molecules encoding
iRNAs. Selection of native polynucleotides is not, however, a
straight-forward process. For example, only a small number of
native polynucleotides in a coleopteran or hemipteran pest will be
effective targets. It cannot be predicted with certainty whether a
particular native polynucleotide can be effectively down-regulated
by nucleic acid molecules of the invention, or whether
down-regulation of a particular native polynucleotide will have a
detrimental effect on the growth, development and/or survival of an
insect pest. The vast majority of native coleopteran and hemipteran
pest polynucleotides, such as ESTs isolated therefrom (for example,
the coleopteran pest polynucleotides listed in U.S. Pat. No.
7,612,194), do not have a detrimental effect on the growth,
development, and/or survival of the pest. Neither is it predictable
which of the native polynucleotides that may have a detrimental
effect on an insect pest are able to be used in recombinant
techniques for expressing nucleic acid molecules complementary to
such native polynucleotides in a host plant and providing the
detrimental effect on the pest upon feeding without causing harm to
the host plant.
[0170] In some embodiments, nucleic acid molecules (e.g., dsRNA
molecules to be provided in the host plant of an insect (e.g.,
coleopteran or hemipteran) pest) are selected to target cDNAs that
encode proteins or parts of proteins essential for pest
development, such as polypeptides involved in metabolic or
catabolic biochemical pathways, cell division, energy metabolism,
digestion, host plant recognition, and the like. As described
herein, ingestion of compositions by a target pest organism
containing one or more dsRNAs, at least one segment of which is
specifically complementary to at least a substantially identical
segment of RNA produced in the cells of the target pest organism,
can result in the death or other inhibition of the target. A
polynucleotide, either DNA or RNA, derived from an insect pest can
be used to construct plant cells resistant to infestation by the
pests. The host plant of the coleopteran and/or hemipteran pest
(e.g., Z. mays or G. max), for example, can be transformed to
contain one or more polynucleotides derived from the coleopteran
and/or hemipteran pest as provided herein. The polynucleotide
transformed into the host may encode one or more RNAs that form
into a dsRNA structure in the cells or biological fluids within the
transformed host, thus making the dsRNA available if/when the pest
forms a nutritional relationship with the transgenic host. This may
result in the suppression of expression of one or more genes in the
cells of the pest, and ultimately death or inhibition of its growth
or development.
[0171] Thus, in some embodiments, a gene is targeted that is
essentially involved in the growth and development of an insect
(e.g., coleopteran or hemipteran) pest. Other target genes for use
in the present invention may include, for example, those that play
important roles in pest movement, migration, growth, development,
infectivity, and establishment of feeding sites. A target gene may
therefore be a housekeeping gene or a transcription factor.
Additionally, a native insect pest polynucleotide for use in the
present invention may also be derived from a homolog (e.g., an
ortholog), of a plant, viral, bacterial or insect gene, the
function of which is known to those of skill in the art, and the
polynucleotide of which is specifically hybridizable with a target
gene in the genome of the target pest. Methods of identifying a
homolog of a gene with a known nucleotide sequence by hybridization
are known to those of skill in the art.
[0172] In some embodiments, the invention provides methods for
obtaining a nucleic acid molecule comprising a polynucleotide for
producing an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA)
molecule. One such embodiment comprises: (a) analyzing one or more
target gene(s) for their expression, function, and phenotype upon
dsRNA-mediated gene suppression in an insect (e.g., coleopteran or
hemipteran) pest; (b) probing a cDNA or gDNA library with a probe
comprising all or a portion of a polynucleotide or a homolog
thereof from a targeted pest that displays an altered (e.g.,
reduced) growth or development phenotype in a dsRNA-mediated
suppression analysis; (c) identifying a DNA clone that specifically
hybridizes with the probe; (d) isolating the DNA clone identified
in step (b); (e) sequencing the cDNA or gDNA fragment that
comprises the clone isolated in step (d), wherein the sequenced
nucleic acid molecule comprises all or a substantial portion of the
RNA or a homolog thereof; and (f) chemically synthesizing all or a
substantial portion of a gene, or an siRNA, miRNA, hpRNA, mRNA,
shRNA, or dsRNA.
[0173] In further embodiments, a method for obtaining a nucleic
acid fragment comprising a polynucleotide for producing a
substantial portion of an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA,
and hpRNA) molecule includes: (a) synthesizing first and second
oligonucleotide primers specifically complementary to a portion of
a native polynucleotide from a targeted insect (e.g., coleopteran
or hemipteran) pest; and (b) amplifying a cDNA or gDNA insert
present in a cloning vector using the first and second
oligonucleotide primers of step (a), wherein the amplified nucleic
acid molecule comprises a substantial portion of a siRNA, miRNA,
hpRNA, mRNA, shRNA, or dsRNA molecule.
[0174] Nucleic acids can be isolated, amplified, or produced by a
number of approaches. For example, an iRNA (e.g., dsRNA, siRNA,
miRNA, shRNA, and hpRNA) molecule may be obtained by PCR
amplification of a target polynucleotide (e.g., a target gene or a
target transcribed non-coding polynucleotide) derived from a gDNA
or cDNA library, or portions thereof. DNA or RNA may be extracted
from a target organism, and nucleic acid libraries may be prepared
therefrom using methods known to those ordinarily skilled in the
art. gDNA or cDNA libraries generated from a target organism may be
used for PCR amplification and sequencing of target genes. A
confirmed PCR product may be used as a template for in vitro
transcription to generate sense and antisense RNA with minimal
promoters. Alternatively, nucleic acid molecules may be synthesized
by any of a number of techniques (See, e.g., Ozaki et al. (1992)
Nucleic Acids Research, 20: 5205-5214; and Agrawal et al. (1990)
Nucleic Acids Research, 18: 5419-5423), including use of an
automated DNA synthesizer (for example, a P.E. Biosystems, Inc.
(Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer), using
standard chemistries, such as phosphoramidite chemistry. See, e.g.,
Beaucage et al. (1992) Tetrahedron, 48: 2223-2311; U.S. Pat. Nos.
4,980,460, 4,725,677, 4,415,732, 4,458,066, and 4,973,679.
Alternative chemistries resulting in non-natural backbone groups,
such as phosphorothioate, phosphoramidate, and the like, can also
be employed.
[0175] An RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA molecule of the
present invention may be produced chemically or enzymatically by
one skilled in the art through manual or automated reactions, or in
vivo in a cell comprising a nucleic acid molecule comprising a
polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA, or
hpRNA molecule. RNA may also be produced by partial or total
organic synthesis--any modified ribonucleotide can be introduced by
in vitro enzymatic or organic synthesis. An RNA molecule may be
synthesized by a cellular RNA polymerase or a bacteriophage RNA
polymerase (e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA
polymerase). Expression constructs useful for the cloning and
expression of polynucleotides are known in the art. See, e.g.,
International PCT Publication No. WO97/32016; and U.S. Pat. Nos.
5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693. RNA
molecules that are synthesized chemically or by in vitro enzymatic
synthesis may be purified prior to introduction into a cell. For
example, RNA molecules can be purified from a mixture by extraction
with a solvent or resin, precipitation, electrophoresis,
chromatography, or a combination thereof. Alternatively, RNA
molecules that are synthesized chemically or by in vitro enzymatic
synthesis may be used with no or a minimum of purification, for
example, to avoid losses due to sample processing. The RNA
molecules may be dried for storage or dissolved in an aqueous
solution. The solution may contain buffers or salts to promote
annealing, and/or stabilization of dsRNA molecule duplex
strands.
[0176] In embodiments, a dsRNA molecule may be formed by a single
self-complementary RNA strand or from two complementary RNA
strands. dsRNA molecules may be synthesized either in vivo or in
vitro. An endogenous RNA polymerase of the cell may mediate
transcription of the one or two RNA strands in vivo, or cloned RNA
polymerase may be used to mediate transcription in vivo or in
vitro. Post-transcriptional inhibition of a target gene in an
insect pest may be host-targeted by specific transcription in an
organ, tissue, or cell type of the host (e.g., by using a
tissue-specific promoter); stimulation of an environmental
condition in the host (e.g., by using an inducible promoter that is
responsive to infection, stress, temperature, and/or chemical
inducers); and/or engineering transcription at a developmental
stage or age of the host (e.g., by using a developmental
stage-specific promoter). RNA strands that form a dsRNA molecule,
whether transcribed in vitro or in vivo, may or may not be
polyadenylated, and may or may not be capable of being translated
into a polypeptide by a cell's translational apparatus.
[0177] D. Recombinant Vectors and Host Cell Transformation
[0178] In some embodiments, the invention also provides a DNA
molecule for introduction into a cell (e.g., a bacterial cell, a
yeast cell, or a plant cell), wherein the DNA molecule comprises a
polynucleotide that, upon expression to RNA and ingestion by an
insect (e.g., coleopteran and/or hemipteran) pest, achieves
suppression of a target gene in a cell, tissue, or organ of the
pest. Thus, some embodiments provide a recombinant nucleic acid
molecule comprising a polynucleotide capable of being expressed as
an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule in a
plant cell to inhibit target gene expression in an insect pest. In
order to initiate or enhance expression, such recombinant nucleic
acid molecules may comprise one or more regulatory elements, which
regulatory elements may be operably linked to the polynucleotide
capable of being expressed as an iRNA. Methods to express a gene
suppression molecule in plants are known, and may be used to
express a polynucleotide of the present invention. See, e.g.,
International PCT Publication No. WO06/073727; and U.S. Patent
Publication No. 2006/0200878 A1)
[0179] In specific embodiments, a recombinant DNA molecule of the
invention may comprise a polynucleotide encoding an RNA that may
form a dsRNA molecule. Such recombinant DNA molecules may encode
RNAs that may form dsRNA molecules capable of inhibiting the
expression of endogenous target gene(s) in an insect (e.g.,
coleopteran and/or hemipteran) pest cell upon ingestion. In many
embodiments, a transcribed RNA may form a dsRNA molecule that may
be provided in a stabilized form; e.g., as a hairpin and stem and
loop structure.
[0180] In some embodiments, one strand of a dsRNA molecule may be
formed by transcription from a polynucleotide which is
substantially homologous to a polynucleotide of any of SEQ ID NO:1
or SEQ ID NO:72; the complement of SEQ ID NO:1 or SEQ ID NO:72; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:1 or
SEQ ID NO:72; the complement of a fragment of at least 15
contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:72; a native
coding sequence of a Diabrotica organism (e.g., WCR) comprising SEQ
ID NO:1 or SEQ ID NO:72; the complement of a native coding sequence
of a Diabrotica organism comprising SEQ ID NO:1 or SEQ ID NO:72; a
native non-coding sequence of a Diabrotica organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:1 or
SEQ ID NO:72; the complement of a native non-coding sequence of a
Diabrotica organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:1 or SEQ ID NO:72; a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Diabrotica
organism comprising any of SEQ ID NO:1 or SEQ ID NO:72; the
complement of a fragment of at least 15 contiguous nucleotides of a
native coding polynucleotide of a Diabrotica organism comprising
SEQ ID NO:1 or SEQ ID NO:72; a fragment of at least 15 contiguous
nucleotides of a native coding polynucleotide of a Diabrotica
organism comprising SEQ ID NO:1 or SEQ ID NO:72; and the complement
of a fragment of at least 15 contiguous nucleotides of a native
coding polynucleotide of a Diabrotica organism comprising SEQ ID
NO:1 or SEQ ID NO:72.
[0181] In some embodiments, one strand of a dsRNA molecule may be
formed by transcription from a polynucleotide that is substantially
homologous to a polynucleotide selected from the group consisting
of SEQ ID NO:83 or SEQ ID NO:84; the complement of SEQ ID NO:83 or
SEQ ID NO:84; a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:83 or SEQ ID NO:84; the complement of a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:83 or SEQ ID NO:84; a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:83 or SEQ ID NO:84; the complement of a native coding sequence
of a hemipteran organism comprising SEQ ID NO:83 or SEQ ID NO:84; a
native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:83 or
SEQ ID NO:84; the complement of a native non-coding sequence of a
hemipteran organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:83 or SEQ ID NO:84; a fragment of at least 15
contiguous nucleotides of a native coding sequence of a hemipteran
organism comprising SEQ ID NO:83 or SEQ ID NO:84; the complement of
a fragment of at least 15 contiguous nucleotides of a native coding
sequence of a hemipteran organism comprising SEQ ID NO:83 or SEQ ID
NO:84; a fragment of at least 15 contiguous nucleotides of a native
non-coding sequence of a hemipteran organism that is transcribed
into a native RNA molecule comprising SEQ ID NO:83 or SEQ ID NO:84;
and the complement of a fragment of at least 15 contiguous
nucleotides of a native non-coding sequence of a hemipteran
organism that is transcribed into a native RNA molecule comprising
SEQ ID NO:83 or SEQ ID NO:84.
[0182] In particular embodiments, a recombinant DNA molecule
encoding an RNA that may form a dsRNA molecule may comprise a
coding region wherein at least two polynucleotides are arranged
such that one polynucleotide is in a sense orientation, and the
other polynucleotide is in an antisense orientation, relative to at
least one promoter, wherein the sense polynucleotide and the
antisense polynucleotide are linked or connected by a spacer of,
for example, from about five (.about.5) to about one thousand
(1000) nucleotides. The spacer may form a loop between the sense
and antisense polynucleotides. The sense polynucleotide or the
antisense polynucleotide may be substantially homologous to a
target gene (e.g., a gene comprising SEQ ID NO:1, SEQ ID NO:72, SEQ
ID NO:83 or SEQ ID NO:84) or fragment thereof. In some embodiments,
however, a recombinant DNA molecule may encode an RNA that may form
a dsRNA molecule without a spacer. In embodiments, a sense coding
polynucleotide and an antisense coding polynucleotide may be
different lengths.
[0183] Polynucleotides identified as having a deleterious effect on
an insect pest or a plant-protective effect with regard to the pest
may be readily incorporated into expressed dsRNA molecules through
the creation of appropriate expression cassettes in a recombinant
nucleic acid molecule of the invention. For example, such
polynucleotides may be expressed as a hairpin with stem and loop
structure by taking a first segment corresponding to a target gene
polynucleotide (e.g., SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or
SEQ ID NO:84 and fragments thereof); linking this polynucleotide to
a second segment spacer region that is not homologous or
complementary to the first segment; and linking this to a third
segment, wherein at least a portion of the third segment is
substantially complementary to the first segment. Such a construct
forms a stem and loop structure by intramolecular base-pairing of
the first segment with the third segment, wherein the loop
structure forms comprising the second segment. See, e.g., U.S.
Patent Publication Nos. 2002/0048814 and 2003/0018993; and
International PCT Publication Nos. WO94/01550 and WO98/05770. A
dsRNA molecule may be generated, for example, in the form of a
double-stranded structure such as a stem-loop structure (e.g.,
hairpin), whereby production of siRNA targeted for a native insect
(e.g., coleopteran and/or hemipteran) pest polynucleotide is
enhanced by co-expression of a fragment of the targeted gene, for
instance on an additional plant expressible cassette, that leads to
enhanced siRNA production, or reduces methylation to prevent
transcriptional gene silencing of the dsRNA hairpin promoter.
[0184] Embodiments of the invention include introduction of a
recombinant nucleic acid molecule of the present invention into a
plant (i.e., transformation) to achieve insect (e.g., coleopteran
and/or hemipteran) pest-inhibitory levels of expression of one or
more iRNA molecules. A recombinant DNA molecule may, for example,
be a vector, such as a linear or a closed circular plasmid. The
vector system may be a single vector or plasmid, or two or more
vectors or plasmids that together contain the total DNA to be
introduced into the genome of a host. In addition, a vector may be
an expression vector. Nucleic acids of the invention can, for
example, be suitably inserted into a vector under the control of a
suitable promoter that functions in one or more hosts to drive
expression of a linked coding polynucleotide or other DNA element.
Many vectors are available for this purpose, and selection of the
appropriate vector will depend mainly on the size of the nucleic
acid to be inserted into the vector and the particular host cell to
be transformed with the vector. Each vector contains various
components depending on its function (e.g., amplification of DNA or
expression of DNA) and the particular host cell with which it is
compatible.
[0185] To impart protection from insect (e.g., coleopteran and/or
hemipteran) pests to a transgenic plant, a recombinant DNA may, for
example, be transcribed into an iRNA molecule (e.g., a RNA molecule
that forms a dsRNA molecule) within the tissues or fluids of the
recombinant plant. An iRNA molecule may comprise a polynucleotide
that is substantially homologous and specifically hybridizable to a
corresponding transcribed polynucleotide within an insect pest that
may cause damage to the host plant species. The pest may contact
the iRNA molecule that is transcribed in cells of the transgenic
host plant, for example, by ingesting cells or fluids of the
transgenic host plant that comprise the iRNA molecule. Thus, in
particular examples, expression of a target gene is suppressed by
the iRNA molecule within coleopteran and/or hemipteran pests that
infest the transgenic host plant. In some embodiments, suppression
of expression of the target gene in a target coleopteran and/or
hemipteran pest may result in the plant being protected from attack
by the pest.
[0186] In order to enable delivery of iRNA molecules to an insect
pest in a nutritional relationship with a plant cell that has been
transformed with a recombinant nucleic acid molecule of the
invention, expression (i.e., transcription) of iRNA molecules in
the plant cell is required. Thus, a recombinant nucleic acid
molecule may comprise a polynucleotide of the invention operably
linked to one or more regulatory elements, such as a heterologous
promoter element that functions in a host cell, such as a bacterial
cell wherein the nucleic acid molecule is to be amplified, and a
plant cell wherein the nucleic acid molecule is to be
expressed.
[0187] Promoters suitable for use in nucleic acid molecules of the
invention include those that are inducible, viral, synthetic, or
constitutive, all of which are well known in the art. Non-limiting
examples describing such promoters include U.S. Pat. No. 6,437,217
(maize RS81 promoter); U.S. Pat. No. 5,641,876 (rice actin
promoter); U.S. Pat. No. 6,426,446 (maize RS324 promoter); U.S.
Pat. No. 6,429,362 (maize PR-1 promoter); U.S. Pat. No. 6,232,526
(maize A3 promoter); U.S. Pat. No. 6,177,611 (constitutive maize
promoters); U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and
5,530,196 (CaMV 35S promoter); U.S. Pat. No. 6,433,252 (maize L3
oleosin promoter); U.S. Pat. No. 6,429,357 (rice actin 2 promoter,
and rice actin 2 intron); U.S. Pat. No. 6,294,714 (light-inducible
promoters); U.S. Pat. No. 6,140,078 (salt-inducible promoters);
U.S. Pat. No. 6,252,138 (pathogen-inducible promoters); U.S. Pat.
No. 6,175,060 (phosphorous deficiency-inducible promoters); U.S.
Pat. No. 6,388,170 (bidirectional promoters); U.S. Pat. No.
6,635,806 (gamma-coixin promoter); and U.S. Patent Publication No.
2009/757,089 (maize chloroplast aldolase promoter). Additional
promoters include the nopaline synthase (NOS) promoter (Ebert et
al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9) and the
octopine synthase (OCS) promoters (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens); the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-24); the
CaMV 35S promoter (Odell et al. (1985) Nature 313:810-2; the
figwort mosaic virus 35S-promoter (Walker et al. (1987) Proc. Natl.
Acad. Sci. USA 84(19):6624-8); the sucrose synthase promoter (Yang
and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-8); the R
gene complex promoter (Chandler et al. (1989) Plant Cell
1:1175-83); the chlorophyll a/b binding protein gene promoter; CaMV
35S (U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and
5,530,196); FMV 35S (U.S. Pat. Nos. 6,051,753, and 5,378,619); a
PC1SV promoter (U.S. Pat. No. 5,850,019); the SCP1 promoter (U.S.
Pat. No. 6,677,503); and AGRtu.nos promoters (GenBank.TM. Accession
No. V00087; Depicker et al. (1982) J. Mol. Appl. Genet. 1:561-73;
Bevan et al. (1983) Nature 304:184-7).
[0188] In particular embodiments, nucleic acid molecules of the
invention comprise a tissue-specific promoter, such as a
root-specific promoter. Root-specific promoters drive expression of
operably-linked coding polynucleotides exclusively or
preferentially in root tissue. Examples of root-specific promoters
are known in the art. See, e.g., U.S. Pat. Nos. 5,110,732;
5,459,252 and 5,837,848; and Opperman et al. (1994) Science
263:221-3; and Hirel et al. (1992) Plant Mol. Biol. 20:207-18. In
some embodiments, a polynucleotide or fragment for coleopteran
and/or hemipteran pest control according to the invention may be
cloned between two root-specific promoters oriented in opposite
transcriptional directions relative to the polynucleotide or
fragment, and which are operable in a transgenic plant cell and
expressed therein to produce RNA molecules in the transgenic plant
cell that subsequently may form dsRNA molecules, as described,
supra. The iRNA molecules expressed in plant tissues may be
ingested by an insect pest so that suppression of target gene
expression is achieved.
[0189] Additional regulatory elements that may optionally be
operably linked to a nucleic acid include 5'UTRs located between a
promoter element and a coding polynucleotide that function as a
translation leader element. The translation leader element is
present in fully-processed mRNA, and it may affect processing of
the primary transcript, and/or RNA stability. Examples of
translation leader elements include maize and petunia heat shock
protein leaders (U.S. Pat. No. 5,362,865), plant virus coat protein
leaders, plant rubisco leaders, and others. See, e.g., Turner and
Foster (1995) Molecular Biotech. 3(3):225-36. Non-limiting examples
of 5'UTRs include GmHsp (U.S. Pat. No. 5,659,122); PhDnaK (U.S.
Pat. No. 5,362,865); AtAnt1; TEV (Carrington and Freed (1990) J.
Virol. 64:1590-7); and AGRtunos (GenBank.TM. Accession No. V00087;
and Bevan et al. (1983) Nature 304:184-7).
[0190] Additional regulatory elements that may optionally be
operably linked to a nucleic acid also include 3' non-translated
elements, 3' transcription termination regions, or polyadenylation
regions. These are genetic elements located downstream of a
polynucleotide, and include polynucleotides that provide
polyadenylation signal, and/or other regulatory signals capable of
affecting transcription or mRNA processing. The polyadenylation
signal functions in plants to cause the addition of polyadenylate
nucleotides to the 3' end of the mRNA precursor. The
polyadenylation element can be derived from a variety of plant
genes, or from T-DNA genes. A non-limiting example of a 3'
transcription termination region is the nopaline synthase 3' region
(nos 3'; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA
80:4803-7). An example of the use of different 3' non-translated
regions is provided in Ingelbrecht et al., (1989) Plant Cell
1:671-80. Non-limiting examples of polyadenylation signals include
one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al.
(1984) EMBO J. 3:1671-9) and AGRtu.nos (GenBank.TM. Accession No.
E01312).
[0191] Some embodiments may include a plant transformation vector
that comprises an isolated and purified DNA molecule comprising at
least one of the above-described regulatory elements operatively
linked to one or more polynucleotides of the present invention.
When expressed, the one or more polynucleotides result in one or
more iRNA molecule(s) comprising a polynucleotide that is
specifically complementary to all or part of a native RNA molecule
in an insect (e.g., coleopteran and/or hemipteran) pest. Thus, the
polynucleotide(s) may comprise a segment encoding all or part of a
polyribonucleotide present within a targeted coleopteran and/or
hemipteran pest RNA transcript, and may comprise inverted repeats
of all or a part of a targeted pest transcript. A plant
transformation vector may contain polynucleotides specifically
complementary to more than one target polynucleotide, thus allowing
production of more than one dsRNA for inhibiting expression of two
or more genes in cells of one or more populations or species of
target insect pests. Segments of polynucleotides specifically
complementary to polynucleotides present in different genes can be
combined into a single composite nucleic acid molecule for
expression in a transgenic plant. Such segments may be contiguous
or separated by a spacer.
[0192] In some embodiments, a plasmid of the present invention
already containing at least one polynucleotide(s) of the invention
can be modified by the sequential insertion of additional
polynucleotide(s) in the same plasmid, wherein the additional
polynucleotide(s) are operably linked to the same regulatory
elements as the original at least one polynucleotide(s). In some
embodiments, a nucleic acid molecule may be designed for the
inhibition of multiple target genes. In some embodiments, the
multiple genes to be inhibited can be obtained from the same insect
(e.g., coleopteran or hemipteran) pest species, which may enhance
the effectiveness of the nucleic acid molecule. In other
embodiments, the genes can be derived from different insect pests,
which may broaden the range of pests against which the agent(s)
is/are effective. When multiple genes are targeted for suppression
or a combination of expression and suppression, a polycistronic DNA
element can be engineered.
[0193] A recombinant nucleic acid molecule or vector of the present
invention may comprise a selectable marker that confers a
selectable phenotype on a transformed cell, such as a plant cell.
Selectable markers may also be used to select for plants or plant
cells that comprise a recombinant nucleic acid molecule of the
invention. The marker may encode biocide resistance, antibiotic
resistance (e.g., kanamycin, Geneticin (G418), bleomycin,
hygromycin, etc.), or herbicide tolerance (e.g., glyphosate, etc.).
Examples of selectable markers include, but are not limited to: a
neo gene which codes for kanamycin resistance and can be selected
for using kanamycin, G418, etc.; a bar gene which codes for
bialaphos resistance; a mutant EPSP synthase gene which encodes
glyphosate tolerance; a nitrilase gene which confers resistance to
bromoxynil; a mutant acetolactate synthase (ALS) gene which confers
imidazolinone or sulfonylurea tolerance; and a methotrexate
resistant DHFR gene. Multiple selectable markers are available that
confer resistance to ampicillin, bleomycin, chloramphenicol,
gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,
phosphinothricin, puromycin, spectinomycin, rifampicin,
streptomycin and tetracycline, and the like. Examples of such
selectable markers are illustrated in, e.g., U.S. Pat. Nos.
5,550,318; 5,633,435; 5,780,708 and 6,118,047.
[0194] A recombinant nucleic acid molecule or vector of the present
invention may also include a screenable marker. Screenable markers
may be used to monitor expression. Exemplary screenable markers
include a .beta.-glucuronidase or uidA gene (GUS) which encodes an
enzyme for which various chromogenic substrates are known
(Jefferson et al. (1987) Plant Mol. Biol. Rep. 5:387-405); an
R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta et
al. (1988) "Molecular cloning of the maize R-nj allele by
transposon tagging with Ac." In 18.sup.th Stadler Genetics
Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp.
263-82); a .beta.-lactamase gene (Sutcliffe et al. (1978) Proc.
Natl. Acad. Sci. USA 75:3737-41); a gene which encodes an enzyme
for which various chromogenic substrates are known (e.g., PADAC, a
chromogenic cephalosporin); a luciferase gene (Ow et al. (1986)
Science 234:856-9); an xylE gene that encodes a catechol
dioxygenase that can convert chromogenic catechols (Zukowski et al.
(1983) Gene 46(2-3):247-55); an amylase gene (Ikatu et al. (1990)
Bio/Technol. 8:241-2); a tyrosinase gene which encodes an enzyme
capable of oxidizing tyrosine to DOPA and dopaquinone which in turn
condenses to melanin (Katz et al. (1983) J. Gen. Microbiol.
129:2703-14); and an .alpha.-galactosidase.
[0195] In some embodiments, recombinant nucleic acid molecules, as
described, supra, may be used in methods for the creation of
transgenic plants and expression of heterologous nucleic acids in
plants to prepare transgenic plants that exhibit reduced
susceptibility to insect (e.g., coleopteran and/or hemipteran)
pests. Plant transformation vectors can be prepared, for example,
by inserting nucleic acid molecules encoding iRNA molecules into
plant transformation vectors and introducing these into plants.
[0196] Suitable methods for transformation of host cells include
any method by which DNA can be introduced into a cell, such as by
transformation of protoplasts (See, e.g., U.S. Pat. No. 5,508,184),
by desiccation/inhibition-mediated DNA uptake (See, e.g., Potrykus
et al. (1985) Mol. Gen. Genet. 199:183-8), by electroporation (See,
e.g., U.S. Pat. No. 5,384,253), by agitation with silicon carbide
fibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), by
Agrobacterium-mediated transformation (See, e.g., U.S. Pat. Nos.
5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and
6,384,301) and by acceleration of DNA-coated particles (See, e.g.,
U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208;
6,399,861; and 6,403,865), etc. Techniques that are particularly
useful for transforming corn are described, for example, in U.S.
Pat. Nos. 7,060,876 and 5,591,616; and International PCT
Publication WO95/06722. Through the application of techniques such
as these, the cells of virtually any species may be stably
transformed. In some embodiments, transforming DNA is integrated
into the genome of the host cell. In the case of multicellular
species, transgenic cells may be regenerated into a transgenic
organism. Any of these techniques may be used to produce a
transgenic plant, for example, comprising one or more nucleic acids
encoding one or more iRNA molecules in the genome of the transgenic
plant.
[0197] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of the plant. The Ti (tumor-inducing)-plasmids
contain a large segment, known as T-DNA, which is transferred to
transformed plants. Another segment of the Ti plasmid, the Vir
region, is responsible for T-DNA transfer. The T-DNA region is
bordered by terminal repeats. In modified binary vectors, the
tumor-inducing genes have been deleted, and the functions of the
Vir region are utilized to transfer foreign DNA bordered by the
T-DNA border elements. The T-region may also contain a selectable
marker for efficient recovery of transgenic cells and plants, and a
multiple cloning site for inserting polynucleotides for transfer
such as a dsRNA encoding nucleic acid.
[0198] Thus, in some embodiments, a plant transformation vector is
derived from a Ti plasmid of A. tumefaciens (See, e.g., U.S. Pat.
Nos. 4,536,475, 4,693,977, 4,886,937, and 5,501,967; and European
Patent No. EP 0 122 791) or a Ri plasmid of A. rhizogenes.
Additional plant transformation vectors include, for example and
without limitation, those described by Herrera-Estrella et al.
(1983) Nature 303:209-13; Bevan et al. (1983) Nature 304:184-7;
Klee et al. (1985) Bio/Technol. 3:637-42; and in European Patent
No. EP 0 120 516, and those derived from any of the foregoing.
Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium
that interact with plants naturally can be modified to mediate gene
transfer to a number of diverse plants. These plant-associated
symbiotic bacteria can be made competent for gene transfer by
acquisition of both a disarmed Ti plasmid and a suitable binary
vector.
[0199] After providing exogenous DNA to recipient cells,
transformed cells are generally identified for further culturing
and plant regeneration. In order to improve the ability to identify
transformed cells, one may desire to employ a selectable or
screenable marker gene, as previously set forth, with the
transformation vector used to generate the transformant. In the
case where a selectable marker is used, transformed cells are
identified within the potentially transformed cell population by
exposing the cells to a selective agent or agents. In the case
where a screenable marker is used, cells may be screened for the
desired marker gene trait.
[0200] Cells that survive the exposure to the selective agent, or
cells that have been scored positive in a screening assay, may be
cultured in media that supports regeneration of plants. In some
embodiments, any suitable plant tissue culture media (e.g., MS and
N6 media) may be modified by including further substances, such as
growth regulators. Tissue may be maintained on a basic medium with
growth regulators until sufficient tissue is available to begin
plant regeneration efforts, or following repeated rounds of manual
selection, until the morphology of the tissue is suitable for
regeneration (e.g., at least 2 weeks), then transferred to media
conducive to shoot formation. Cultures are transferred periodically
until sufficient shoot formation has occurred. Once shoots are
formed, they are transferred to media conducive to root formation.
Once sufficient roots are formed, plants can be transferred to soil
for further growth and maturation.
[0201] To confirm the presence of a nucleic acid molecule of
interest (for example, a DNA encoding one or more iRNA molecules
that inhibit target gene expression in a coleopteran and/or
hemipteran pest) in the regenerating plants, a variety of assays
may be performed. Such assays include, for example: molecular
biological assays, such as Southern and northern blotting, PCR, and
nucleic acid sequencing; biochemical assays, such as detecting the
presence of a protein product, e.g., by immunological means (ELISA
and/or western blots) or by enzymatic function; plant part assays,
such as leaf or root assays; and analysis of the phenotype of the
whole regenerated plant.
[0202] Integration events may be analyzed, for example, by PCR
amplification using, e.g., oligonucleotide primers specific for a
nucleic acid molecule of interest. PCR genotyping is understood to
include, but not be limited to, polymerase-chain reaction (PCR)
amplification of gDNA derived from isolated host plant callus
tissue predicted to contain a nucleic acid molecule of interest
integrated into the genome, followed by standard cloning and
sequence analysis of PCR amplification products. Methods of PCR
genotyping have been well described (for example, Rios, G. et al.
(2002) Plant J. 32:243-53) and may be applied to gDNA derived from
any plant species (e.g., Z. mays or G. max) or tissue type,
including cell cultures.
[0203] A transgenic plant formed using Agrobacterium-dependent
transformation methods typically contains a single recombinant DNA
inserted into one chromosome. The polynucleotide of the single
recombinant DNA is referred to as a "transgenic event" or
"integration event". Such transgenic plants are heterozygous for
the inserted exogenous polynucleotide. In some embodiments, a
transgenic plant homozygous with respect to a transgene may be
obtained by sexually mating (selfing) an independent segregant
transgenic plant that contains a single exogenous gene to itself,
for example a T.sub.0 plant, to produce T.sub.1 seed. One fourth of
the T.sub.1 seed produced will be homozygous with respect to the
transgene. Germinating T.sub.1 seed results in plants that can be
tested for heterozygosity, typically using an SNP assay or a
thermal amplification assay that allows for the distinction between
heterozygotes and homozygotes (i.e., a zygosity assay).
[0204] In particular embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9
or 10 or more different iRNA molecules are produced in a plant cell
that have an insect (e.g., coleopteran and/or hemipteran)
pest-inhibitory effect. The iRNA molecules (e.g., dsRNA molecules)
may be expressed from multiple nucleic acids introduced in
different transformation events, or from a single nucleic acid
introduced in a single transformation event. In some embodiments, a
plurality of iRNA molecules are expressed under the control of a
single promoter. In other embodiments, a plurality of iRNA
molecules are expressed under the control of multiple promoters.
Single iRNA molecules may be expressed that comprise multiple
polynucleotides that are each homologous to different loci within
one or more insect pests (for example, the loci defined by SEQ ID
NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84), both in
different populations of the same species of insect pest, or in
different species of insect pests.
[0205] In addition to direct transformation of a plant with a
recombinant nucleic acid molecule, transgenic plants can be
prepared by crossing a first plant having at least one transgenic
event with a second plant lacking such an event. For example, a
recombinant nucleic acid molecule comprising a polynucleotide that
encodes an iRNA molecule may be introduced into a first plant line
that is amenable to transformation to produce a transgenic plant,
which transgenic plant may be crossed with a second plant line to
introgress the polynucleotide that encodes the iRNA molecule into
the second plant line.
[0206] The invention also includes commodity products containing
one or more of the sequences of the present invention. Particular
embodiments include commodity products produced from a recombinant
plant or seed containing one or more of the nucleotide sequences of
the present invention. A commodity product containing one or more
of the sequences of the present invention is intended to include,
but not be limited to, meals, oils, crushed or whole grains or
seeds of a plant, or any food or animal feed product comprising any
meal, oil, or crushed or whole grain of a recombinant plant or seed
containing one or more of the sequences of the present invention.
The detection of one or more of the sequences of the present
invention in one or more commodity or commodity products
contemplated herein is de facto evidence that the commodity or
commodity product is produced from a transgenic plant designed to
express one or more of the nucleotides sequences of the present
invention for the purpose of controlling coleopteran and/or
hemipteran plant pests using dsRNA-mediated gene suppression
methods.
[0207] In some aspects, seeds and commodity products produced by
transgenic plants derived from transformed plant cells are
included, wherein the seeds or commodity products comprise a
detectable amount of a nucleic acid of the invention. In some
embodiments, such commodity products may be produced, for example,
by obtaining transgenic plants and preparing food or feed from
them. Commodity products comprising one or more of the
polynucleotides of the invention includes, for example and without
limitation: meals, oils, crushed or whole grains or seeds of a
plant, and any food product comprising any meal, oil, or crushed or
whole grain of a recombinant plant or seed comprising one or more
of the nucleic acids of the invention. The detection of one or more
of the polynucleotides of the invention in one or more commodity or
commodity products is de facto evidence that the commodity or
commodity product is produced from a transgenic plant designed to
express one or more of the iRNA molecules of the invention for the
purpose of controlling insect (e.g., coleopteran and/or hemipteran)
pests.
[0208] In some embodiments, a transgenic plant or seed comprising a
nucleic acid molecule of the invention also may comprise at least
one other transgenic event in its genome, including without
limitation: a transgenic event from which is a transcribed iRNA
molecule targeting a locus in an insect pest other than the one
defined by SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or SEQ ID NO:84,
such as, for example, one or more loci selected from the group
consisting of Caf1-180 (U.S. Patent Application Publication No.
2012/0174258), VatpaseC (U.S. Patent Application Publication No.
2012/0174259), Rho1 (U.S. Patent Application Publication No.
2012/0174260), VatpaseH (U.S. Patent Application Publication No.
2012/0198586), PPI-87B (U.S. Patent Application Publication No.
2013/0091600), RPA70 (U.S. Patent Application Publication No.
2013/0091601), and RPS6 (U.S. Patent Application Publication No.
2013/0097730); a transgenic event from which is transcribed an iRNA
molecule targeting a gene in an organism other than a coleopteran
and/or hemipteran pest (e.g., a plant-parasitic nematode); a gene
encoding an insecticidal protein (e.g., a Bacillus thuringiensis,
Alcaligenes spp. (e.g., U.S. Patent Application Publication No.
2014/0033361) or Pseudomonas spp. (e.g., PCT Application
Publication No. WO2015038734) insecticidal protein); an herbicide
tolerance gene (e.g., a gene providing tolerance to glyphosate);
and a gene contributing to a desirable phenotype in the transgenic
plant, such as increased yield, altered fatty acid metabolism, or
restoration of cytoplasmic male sterility). In particular
embodiments, polynucleotides encoding iRNA molecules of the
invention may be combined with other insect control and disease
traits in a plant to achieve desired traits for enhanced control of
plant disease and insect damage. Combining insect control traits
that employ distinct modes-of-action may provide protected
transgenic plants with superior durability over plants harboring a
single control trait, for example, because of the reduced
probability that resistance to the trait(s) will develop in the
field.
V. Target Gene Suppression in a Coleopteran and/or Hemipteran
Pest
[0209] A. Overview
[0210] In some embodiments of the invention, at least one nucleic
acid molecule useful for the control of coleopteran and/or
hemipteran pests may be provided to a coleopteran and/or hemipteran
pest, wherein the nucleic acid molecule leads to RNAi-mediated gene
silencing in the pest(s). In particular embodiments, an iRNA
molecule (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) may be
provided to the coleopteran and/or hemipteran host. In some
embodiments, a nucleic acid molecule useful for the control of
coleopteran and/or hemipteran pests may be provided to a pest by
contacting the nucleic acid molecule with the pest. In these and
further embodiments, a nucleic acid molecule useful for the control
of coleopteran and/or hemipteran pests may be provided in a feeding
substrate of the pest, for example, a nutritional composition. In
these and further embodiments, a nucleic acid molecule useful for
the control of a coleopteran and/or hemipteran pest may be provided
through ingestion of plant material comprising the nucleic acid
molecule that is ingested by the pest. In certain embodiments, the
nucleic acid molecule is present in plant material through
expression of a recombinant nucleic acid introduced into the plant
material, for example, by transformation of a plant cell with a
vector comprising the recombinant nucleic acid and regeneration of
a plant material or whole plant from the transformed plant
cell.
[0211] B. RNAi-Mediated Target Gene Suppression
[0212] In embodiments, the invention provides iRNA molecules (e.g.,
dsRNA, siRNA, miRNA, shRNA, and hpRNA) that may be designed to
target essential native polynucleotides (e.g., essential genes) in
the transcriptome of an insect pest (for example, a coleopteran
(e.g., WCR or NCR) or hemipteran (e.g., BSB) pest), for example by
designing an iRNA molecule that comprises at least one strand
comprising a polynucleotide that is specifically complementary to
the target polynucleotide. The sequence of an iRNA molecule so
designed may be identical to that of the target polynucleotide, or
may incorporate mismatches that do not prevent specific
hybridization between the iRNA molecule and its target
polynucleotide.
[0213] iRNA molecules of the invention may be used in methods for
gene suppression in an insect (e.g., coleopteran and/or hemipteran)
pest, thereby reducing the level or incidence of damage caused by
the pest on a plant (for example, a protected transformed plant
comprising an iRNA molecule). As used herein the term "gene
suppression" refers to any of the well-known methods for reducing
the levels of protein produced as a result of gene transcription to
mRNA and subsequent translation of the mRNA, including the
reduction of protein expression from a gene or a coding
polynucleotide including post-transcriptional inhibition of
expression and transcriptional suppression. Post-transcriptional
inhibition is mediated by specific homology between all or a part
of an mRNA transcribed from a gene targeted for suppression and the
corresponding iRNA molecule used for suppression. Additionally,
post-transcriptional inhibition refers to the substantial and
measurable reduction of the amount of mRNA available in the cell
for binding by ribosomes.
[0214] In embodiments wherein an iRNA molecule is a dsRNA molecule,
the dsRNA molecule may be cleaved by the enzyme, DICER, into short
siRNA molecules (approximately 20 nucleotides in length). The
double-stranded siRNA molecule generated by DICER activity upon the
dsRNA molecule may be separated into two single-stranded siRNAs;
the "passenger strand" and the "guide strand". The passenger strand
may be degraded, and the guide strand may be incorporated into
RISC. Post-transcriptional inhibition occurs by specific
hybridization of the guide strand with a specifically complementary
polynucleotide of an mRNA molecule, and subsequent cleavage by the
enzyme, Argonaute (catalytic component of the RISC complex).
[0215] In embodiments of the invention, any form of iRNA molecule
may be used. Those of skill in the art will understand that dsRNA
molecules typically are more stable during preparation and during
the step of providing the iRNA molecule to a cell than are
single-stranded RNA molecules, and are typically also more stable
in a cell. Thus, while siRNA and miRNA molecules, for example, may
be equally effective in some embodiments, a dsRNA molecule may be
chosen due to its stability.
[0216] In particular embodiments, a nucleic acid molecule is
provided that comprises a polynucleotide, which polynucleotide may
be expressed in vitro to produce an iRNA molecule that is
substantially homologous to a nucleic acid molecule encoded by a
polynucleotide within the genome of an insect (e.g., coleopteran
and/or hemipteran) pest. In certain embodiments, the in vitro
transcribed iRNA molecule may be a stabilized dsRNA molecule that
comprises a stem-loop structure. After an insect pest contacts the
in vitro transcribed iRNA molecule, post-transcriptional inhibition
of a target gene in the pest (for example, an essential gene) may
occur.
[0217] In some embodiments of the invention, expression of a
nucleic acid molecule comprising at least 15 contiguous nucleotides
(e.g., at least 19 contiguous nucleotides) of a polynucleotide are
used in a method for post-transcriptional inhibition of a target
gene in an insect (e.g., coleopteran and/or hemipteran) pest,
wherein the polynucleotide is selected from the group consisting
of: SEQ ID NO:1 or SEQ ID NO:72; the complement of SEQ ID NO:1 or
SEQ ID NO:72; a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:1 or SEQ ID NO:72; the complement of a fragment of at
least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:72; a
native coding polynucleotide of a Diabrotica organism comprising
SEQ ID NO:1 or SEQ ID NO:72; the complement of an RNA expressed
from a native coding polynucleotide of a Diabrotica organism
comprising SEQ ID NO:1 or SEQ ID NO:72; an RNA expressed from a
native coding polynucleotide of a Diabrotica organism comprising
SEQ ID NO:1 or SEQ ID NO:72; the complement of an RNA expressed
from a native coding polynucleotide of a Diabrotica organism
comprising SEQ ID NO:1 or SEQ ID NO:72; the complement of an RNA
expressed from a native coding polynucleotide of a Diabrotica
organism that is transcribed into a native RNA molecule comprising
SEQ ID NO:1 or SEQ ID NO:72; a fragment of at least 15 contiguous
nucleotides of a native coding sequence of a Diabrotica organism
(e.g., WCR) comprising SEQ ID NO:1 or SEQ ID NO:72; the complement
of a fragment of at least 15 contiguous nucleotides of a native
coding sequence of a Diabrotica organism comprising SEQ ID NO:1 or
SEQ ID NO:72; a fragment of at least 15 contiguous nucleotides of a
native non-coding sequence of a Diabrotica organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:1 or
SEQ ID NO:72; and the complement of a fragment of at least 15
contiguous nucleotides of a native non-coding sequence of a
Diabrotica organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:1 or SEQ ID NO:72. In certain embodiments,
expression of a nucleic acid molecule that is at least about 80%
identical (e.g., 79%, about 80%, about 81%, about 82%, about 83%,
about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, about 100%, and 100%)
with any of the foregoing may be used.
[0218] In certain embodiments of the invention, expression of a
nucleic acid molecule comprising at least 15 contiguous nucleotides
of a nucleotide sequence is used in a method for
post-transcriptional inhibition of a target gene in a hemipteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:83 or SEQ ID NO:84; the complement of SEQ
ID NO:83 or SEQ ID NO:84; a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:83 or SEQ ID NO:84; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:83 or
SEQ ID NO:84; a native coding sequence of a hemipteran organism SEQ
ID NO:83 or SEQ ID NO:84; the complement of a native coding
sequence of a hemipteran organism comprising SEQ ID NO:83 or SEQ ID
NO:84; a native non-coding sequence of a hemipteran organism that
is transcribed into a native RNA molecule comprising SEQ ID NO:83
or SEQ ID NO:84; the complement of a native non-coding sequence of
a hemipteran organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:83 or SEQ ID NO:84; the complement of
a native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:83 or
SEQ ID NO:84; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:83 or SEQ ID NO:84; the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a hemipteran
organism comprising SEQ ID NO:83 or SEQ ID NO:84; a fragment of at
least 15 contiguous nucleotides of a native non-coding sequence of
a hemipteran organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:83 or SEQ ID NO:84; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:83 or
SEQ ID NO:84. In certain embodiments, expression of a nucleic acid
molecule that is at least 80% identical (e.g., 80%, about 81%,
about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,
about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
about 100%, and 100%) with any of the foregoing may be used. In
these and further embodiments, a nucleic acid molecule may be
expressed that specifically hybridizes to an RNA molecule present
in at least one cell of an insect (e.g., coleopteran and/or
hemipteran) pest.
[0219] In some embodiments, expression of at least one nucleic acid
molecule comprising at least 15 contiguous nucleotides of a
nucleotide sequence may be used in a method for
post-transcriptional inhibition of a target gene in a coleopteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; the
complement of a fragment of at least 15 contiguous nucleotides of
SEQ ID NO:1; a native coding sequence of a Diabrotica organism
(e.g., WCR) comprising SEQ ID NO:1; the complement of a native
coding sequence of a Diabrotica organism (e.g., WCR) comprising SEQ
ID NO:1; a native non-coding sequence of a Diabrotica organism that
is transcribed into a native RNA molecule comprising SEQ ID NO:1;
the complement of a native non-coding sequence of a Diabrotica
organism that is transcribed into a native RNA molecule comprising
SEQ ID NO:1; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a Diabrotica organism (e.g., WCR)
comprising SEQ ID NO:1; the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a Diabrotica
organism comprising SEQ ID NO:1; a fragment of at least 15
contiguous nucleotides of a native non-coding sequence of a
Diabrotica organism that is transcribed into a native RNA molecule
comprising SEQ ID NO:1; and the complement of a fragment of at
least 15 contiguous nucleotides of a native non-coding sequence of
a Diabrotica organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:1. In certain embodiments, expression
of a nucleic acid molecule that is at least 80% identical (e.g.,
80%, about 81%, about 82%, about 83%, about 84%, about 85%, about
86%, about 87%, about 88%, about 89%, about 90%, about 91%, about
92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
98%, about 99%, about 100%, and 100%) with any of the foregoing may
be used. In these and further embodiments, a nucleic acid molecule
may be expressed that specifically hybridizes to an RNA molecule
present in at least one cell of a coleopteran pest. In particular
examples, such a nucleic acid molecule may comprise a nucleotide
sequence comprising SEQ ID NO:1.
[0220] In particular embodiments of the invention, expression of a
nucleic acid molecule comprising at least 15 contiguous nucleotides
of a nucleotide sequence is used in a method for
post-transcriptional inhibition of a target gene in a hemipteran
pest, wherein the nucleotide sequence is selected from the group
consisting of: SEQ ID NO:83 or SEQ ID NO:84; the complement of SEQ
ID NO:83 or SEQ ID NO:84; a fragment of at least 15 contiguous
nucleotides of SEQ ID NO:83 or SEQ ID NO:84; the complement of a
fragment of at least 15 contiguous nucleotides of SEQ ID NO:83 or
SEQ ID NO:84; a native coding sequence of a hemipteran organism SEQ
ID NO:83 or SEQ ID NO:84; the complement of a native coding
sequence of a hemipteran organism comprising SEQ ID NO:83 or SEQ ID
NO:84; a native non-coding sequence of a hemipteran organism that
is transcribed into a native RNA molecule comprising SEQ ID NO:83
or SEQ ID NO:84; the complement of a native non-coding sequence of
a hemipteran organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:83 or SEQ ID NO:84; the complement of
a native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:83 or
SEQ ID NO:84; a fragment of at least 15 contiguous nucleotides of a
native coding sequence of a hemipteran organism comprising SEQ ID
NO:83 or SEQ ID NO:84; the complement of a fragment of at least 15
contiguous nucleotides of a native coding sequence of a hemipteran
organism comprising SEQ ID NO:83 or SEQ ID NO:84; a fragment of at
least 15 contiguous nucleotides of a native non-coding sequence of
a hemipteran organism that is transcribed into a native RNA
molecule comprising SEQ ID NO:83 or SEQ ID NO:84; and the
complement of a fragment of at least 15 contiguous nucleotides of a
native non-coding sequence of a hemipteran organism that is
transcribed into a native RNA molecule comprising SEQ ID NO:83 or
SEQ ID NO:84. In certain embodiments, expression of a nucleic acid
molecule that is at least 80% identical (e.g., 80%, about 81%,
about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,
about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,
about 100%, and 100%) with any of the foregoing may be used. In
these and further embodiments, a nucleic acid molecule may be
expressed that specifically hybridizes to an RNA molecule present
in at least one cell of a hemipteran pest. In particular examples,
such a nucleic acid molecule may comprise a nucleotide sequence
comprising SEQ ID NO:83 or SEQ ID NO:84.
[0221] It is an important feature of some embodiments herein that
the RNAi post-transcriptional inhibition system is able to tolerate
sequence variations among target genes that might be expected due
to genetic mutation, strain polymorphism, or evolutionary
divergence. The introduced nucleic acid molecule may not need to be
absolutely homologous to either a primary transcription product or
a fully-processed mRNA of a target gene, so long as the introduced
nucleic acid molecule is specifically hybridizable to either a
primary transcription product or a fully-processed mRNA of the
target gene. Moreover, the introduced nucleic acid molecule may not
need to be full-length, relative to either a primary transcription
product or a fully processed mRNA of the target gene.
[0222] Inhibition of a target gene using the iRNA technology of the
present invention is sequence-specific; i.e., polynucleotides
substantially homologous to the iRNA molecule(s) are targeted for
genetic inhibition. In some embodiments, an RNA molecule comprising
a polynucleotide with a nucleotide sequence that is identical to
that of a portion of a target gene may be used for inhibition. In
these and further embodiments, an RNA molecule comprising a
polynucleotide with one or more insertion, deletion, and/or point
mutations relative to a target polynucleotide may be used. In
particular embodiments, an iRNA molecule and a portion of a target
gene may share, for example, at least from about 80%, at least from
about 81%, at least from about 82%, at least from about 83%, at
least from about 84%, at least from about 85%, at least from about
86%, at least from about 87%, at least from about 88%, at least
from about 89%, at least from about 90%, at least from about 91%,
at least from about 92%, at least from about 93%, at least from
about 94%, at least from about 95%, at least from about 96%, at
least from about 97%, at least from about 98%, at least from about
99%, at least from about 100%, and 100% sequence identity.
Alternatively, the duplex region of a dsRNA molecule may be
specifically hybridizable with a portion of a target gene
transcript. In specifically hybridizable molecules, a less than
full length polynucleotide exhibiting a greater homology
compensates for a longer, less homologous polynucleotide. The
length of the polynucleotide of a duplex region of a dsRNA molecule
that is identical to a portion of a target gene transcript may be
at least about 25, 50, 100, 200, 300, 400, 500, or at least about
1000 bases. In some embodiments, a polynucleotide of greater than
20-100 nucleotides may be used. In particular embodiments, a
polynucleotide of greater than about 200-300 nucleotides may be
used. In particular embodiments, a polynucleotide of greater than
about 500-1000 nucleotides may be used, depending on the size of
the target gene.
[0223] In certain embodiments, expression of a target gene in a
pest (e.g., coleopteran or hemipteran) pest may be inhibited by at
least 10%; at least 33%; at least 50%; or at least 80% within a
cell of the pest, such that a significant inhibition takes place.
Significant inhibition refers to inhibition over a threshold that
results in a detectable phenotype (e.g., cessation of growth,
cessation of feeding, cessation of development, induced mortality,
etc.), or a detectable decrease in RNA and/or gene product
corresponding to the target gene being inhibited. Although, in
certain embodiments of the invention, inhibition occurs in
substantially all cells of the pest, in other embodiments
inhibition occurs only in a subset of cells expressing the target
gene.
[0224] In some embodiments, transcriptional suppression is mediated
by the presence in a cell of a dsRNA molecule exhibiting
substantial sequence identity to a promoter DNA or the complement
thereof to effect what is referred to as "promoter trans
suppression." Gene suppression may be effective against target
genes in an insect pest that may ingest or contact such dsRNA
molecules, for example, by ingesting or contacting plant material
containing the dsRNA molecules. dsRNA molecules for use in promoter
trans suppression may be specifically designed to inhibit or
suppress the expression of one or more homologous or complementary
polynucleotides in the cells of the insect pest.
Post-transcriptional gene suppression by antisense or sense
oriented RNA to regulate gene expression in plant cells is
disclosed in U.S. Pat. Nos. 5,107,065; 5,759,829; 5,283,184; and
5,231,020.
[0225] C. Expression of iRNA Molecules Provided to an Insect
Pest
[0226] Expression of iRNA molecules for RNAi-mediated gene
inhibition in an insect (e.g., coleopteran and/or hemipteran) pest
may be carried out in any one of many in vitro or in vivo formats.
The iRNA molecules may then be provided to an insect pest, for
example, by contacting the iRNA molecules with the pest, or by
causing the pest to ingest or otherwise internalize the iRNA
molecules. Some embodiments include transformed host plants of a
coleopteran and/or hemipteran pest, transformed plant cells, and
progeny of transformed plants. The transformed plant cells and
transformed plants may be engineered to express one or more of the
iRNA molecules, for example, under the control of a heterologous
promoter, to provide a pest-protective effect. Thus, when a
transgenic plant or plant cell is consumed by an insect pest during
feeding, the pest may ingest iRNA molecules expressed in the
transgenic plants or cells. The polynucleotides of the present
invention may also be introduced into a wide variety of prokaryotic
and eukaryotic microorganism hosts to produce iRNA molecules. The
term "microorganism" includes prokaryotic and eukaryotic species,
such as bacteria and fungi.
[0227] Modulation of gene expression may include partial or
complete suppression of such expression. In another embodiment, a
method for suppression of gene expression in an insect (e.g.,
coleopteran and/or hemipteran) pest comprises providing in the
tissue of the host of the pest a gene-suppressive amount of at
least one dsRNA molecule formed following transcription of a
polynucleotide as described herein, at least one segment of which
is complementary to an mRNA within the cells of the insect pest. A
dsRNA molecule, including its modified form such as an siRNA,
miRNA, shRNA, or hpRNA molecule, ingested by an insect pest may be
at least from about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about
100% identical to an RNA molecule transcribed from a COPI beta DNA
molecule, for example, comprising a polynucleotide selected from
the group consisting of SEQ ID NO:1, SEQ ID NO:72, SEQ ID NO:83 or
SEQ ID NO:84. Isolated and substantially purified nucleic acid
molecules including, but not limited to, non-naturally occurring
polynucleotides and recombinant DNA constructs for providing dsRNA
molecules are therefore provided, which suppress or inhibit the
expression of an endogenous coding polynucleotide or a target
coding polynucleotide in an insect pest when introduced
thereto.
[0228] Particular embodiments provide a delivery system for the
delivery of iRNA molecules for the post-transcriptional inhibition
of one or more target gene(s) in an insect (e.g., coleopteran
and/or hemipteran) plant pest and control of a population of the
plant pest. In some embodiments, the delivery system comprises
ingestion of a host transgenic plant cell or contents of the host
cell comprising RNA molecules transcribed in the host cell. In
these and further embodiments, a transgenic plant cell or a
transgenic plant is created that contains a recombinant DNA
construct providing a stabilized dsRNA molecule of the invention.
Transgenic plant cells and transgenic plants comprising nucleic
acids encoding a particular iRNA molecule may be produced by
employing recombinant DNA technologies (which basic technologies
are well-known in the art) to construct a plant transformation
vector comprising a polynucleotide encoding an iRNA molecule of the
invention (e.g., a stabilized dsRNA molecule); to transform a plant
cell or plant; and to generate the transgenic plant cell or the
transgenic plant that contains the transcribed iRNA molecule.
[0229] To impart protection from insect (e.g., coleopteran and/or
hemipteran) pests to a transgenic plant, a recombinant DNA molecule
may, for example, be transcribed into an iRNA molecule, such as a
dsRNA molecule, an siRNA molecule, an miRNA molecule, an shRNA
molecule, or an hpRNA molecule. In some embodiments, an RNA
molecule transcribed from a recombinant DNA molecule may form a
dsRNA molecule within the tissues or fluids of the recombinant
plant. Such a dsRNA molecule may be comprised in part of a
polynucleotide that is identical to a corresponding polynucleotide
transcribed from a DNA within an insect pest of a type that may
infest the host plant. Expression of a target gene within the pest
is suppressed by the dsRNA molecule, and the suppression of
expression of the target gene in the pest results in the transgenic
plant being resistant to the pest. The modulatory effects of dsRNA
molecules have been shown to be applicable to a variety of genes
expressed in pests, including, for example, endogenous genes
responsible for cellular metabolism or cellular transformation,
including house-keeping genes; transcription factors;
molting-related genes; and other genes which encode polypeptides
involved in cellular metabolism or normal growth and
development.
[0230] For transcription from a transgene in vivo or an expression
construct, a regulatory region (e.g., promoter, enhancer, silencer,
and polyadenylation signal) may be used in some embodiments to
transcribe the RNA strand (or strands). Therefore, in some
embodiments, as set forth, supra, a polynucleotide for use in
producing iRNA molecules may be operably linked to one or more
promoter elements functional in a plant host cell. The promoter may
be an endogenous promoter, normally resident in the host genome.
The polynucleotide of the present invention, under the control of
an operably linked promoter element, may further be flanked by
additional elements that advantageously affect its transcription
and/or the stability of a resulting transcript. Such elements may
be located upstream of the operably linked promoter, downstream of
the 3' end of the expression construct, and may occur both upstream
of the promoter and downstream of the 3' end of the expression
construct.
[0231] Some embodiments provide methods for reducing the damage to
a host plant (e.g., a corn plant) caused by an insect (e.g.,
coleopteran and/or hemipteran) pest that feeds on the plant,
wherein the method comprises providing in the host plant a
transformed plant cell expressing at least one nucleic acid
molecule of the invention, wherein the nucleic acid molecule(s)
functions upon being taken up by the pest(s) to inhibit the
expression of a target polynucleotide within the pest(s), which
inhibition of expression results in mortality and/or reduced growth
of the pest(s), thereby reducing the damage to the host plant
caused by the pest(s). In some embodiments, the nucleic acid
molecule(s) comprise dsRNA molecules. In these and further
embodiments, the nucleic acid molecule(s) comprise dsRNA molecules
that each comprise more than one polynucleotide that is
specifically hybridizable to a nucleic acid molecule expressed in a
coleopteran and/or hemipteran pest cell. In some embodiments, the
nucleic acid molecule(s) consist of one polynucleotide that is
specifically hybridizable to a nucleic acid molecule expressed in
an insect pest cell.
[0232] In some embodiments, a method for increasing the yield of a
corn crop is provided, wherein the method comprises introducing
into a corn plant at least one nucleic acid molecule of the
invention; cultivating the corn plant to allow the expression of an
iRNA molecule comprising the nucleic acid, wherein expression of an
iRNA molecule comprising the nucleic acid inhibits insect (e.g.,
coleopteran and/or hemipteran) pest damage and/or growth, thereby
reducing or eliminating a loss of yield due to pest infestation. In
some embodiments, the iRNA molecule is a dsRNA molecule. In these
and further embodiments, the nucleic acid molecule(s) comprise
dsRNA molecules that each comprise more than one polynucleotide
that is specifically hybridizable to a nucleic acid molecule
expressed in an insect pest cell. In some examples, the nucleic
acid molecule(s) comprises a polynucleotide that is specifically
hybridizable to a nucleic acid molecule expressed in a coleopteran
and/or hemipteran pest cell.
[0233] In some embodiments, a method for modulating the expression
of a target gene in an insect (e.g., coleopteran and/or hemipteran)
pest is provided, the method comprising: transforming a plant cell
with a vector comprising a polynucleotide encoding at least one
iRNA molecule of the invention, wherein the polynucleotide is
operatively-linked to a promoter and a transcription termination
element; culturing the transformed plant cell under conditions
sufficient to allow for development of a plant cell culture
including a plurality of transformed plant cells; selecting for
transformed plant cells that have integrated the polynucleotide
into their genomes; screening the transformed plant cells for
expression of an iRNA molecule encoded by the integrated
polynucleotide; selecting a transgenic plant cell that expresses
the iRNA molecule; and feeding the selected transgenic plant cell
to the insect pest. Plants may also be regenerated from transformed
plant cells that express an iRNA molecule encoded by the integrated
nucleic acid molecule. In some embodiments, the iRNA molecule is a
dsRNA molecule. In these and further embodiments, the nucleic acid
molecule(s) comprise dsRNA molecules that each comprise more than
one polynucleotide that is specifically hybridizable to a nucleic
acid molecule expressed in an insect pest cell. In some examples,
the nucleic acid molecule(s) comprises a polynucleotide that is
specifically hybridizable to a nucleic acid molecule expressed in a
coleopteran and/or hemipteran pest cell.
[0234] iRNA molecules of the invention can be incorporated within
the seeds of a plant species (e.g., corn), either as a product of
expression from a recombinant gene incorporated into a genome of
the plant cells, or as incorporated into a coating or seed
treatment that is applied to the seed before planting. A plant cell
comprising a recombinant gene is considered to be a transgenic
event. Also included in embodiments of the invention are delivery
systems for the delivery of iRNA molecules to insect (e.g.,
coleopteran and/or hemipteran) pests. For example, the iRNA
molecules of the invention may be directly introduced into the
cells of a pest(s). Methods for introduction may include direct
mixing of iRNA with plant tissue from a host for the insect
pest(s), as well as application of compositions comprising iRNA
molecules of the invention to host plant tissue. For example, iRNA
molecules may be sprayed onto a plant surface. Alternatively, an
iRNA molecule may be expressed by a microorganism, and the
microorganism may be applied onto the plant surface, or introduced
into a root or stem by a physical means such as an injection. As
discussed, supra, a transgenic plant may also be genetically
engineered to express at least one iRNA molecule in an amount
sufficient to kill the insect pests known to infest the plant. iRNA
molecules produced by chemical or enzymatic synthesis may also be
formulated in a manner consistent with common agricultural
practices, and used as spray-on products for controlling plant
damage by an insect pest. The formulations may include the
appropriate adjuvants (e.g., stickers and wetters) required for
efficient foliar coverage, as well as UV protectants to protect
iRNA molecules (e.g., dsRNA molecules) from UV damage. Such
additives are commonly used in the bioinsecticide industry, and are
well known to those skilled in the art. Such applications may be
combined with other spray-on insecticide applications (biologically
based or otherwise) to enhance plant protection from the pests.
[0235] All references, including publications, patents, and patent
applications, cited herein are hereby incorporated by reference to
the extent they are not inconsistent with the explicit details of
this disclosure, and are so incorporated to the same extent as if
each reference were individually and specifically indicated to be
incorporated by reference and were set forth in its entirety
herein. The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0236] The following EXAMPLES are provided to illustrate certain
particular features and/or aspects. These EXAMPLES should not be
construed to limit the disclosure to the particular features or
aspects described.
EXAMPLES
Example 1
Insect Diet Bioassays
[0237] Sample Preparation and Bioassays
[0238] A number of dsRNA molecules (including those corresponding
to COPI beta reg1 (SEQ ID NO:3), COPI beta reg2 (SEQ ID NO:68),
COPI beta reg3 (SEQ ID NO:69), COPI beta ver1 (SEQ ID NO:70), COPI
beta ver2 (SEQ ID NO:71), COPI beta var1 (SEQ ID NO:72) were
synthesized and purified using a MEGASCRIPT.RTM. RNAi kit. The
purified dsRNA molecules were prepared in TE buffer, and all
bioassays contained a control treatment consisting of this buffer,
which served as a background check for mortality or growth
inhibition of WCR (Diabrotica virgifera virgifera LeConte). The
concentrations of dsRNA molecules in the bioassay buffer were
measured using a NANODROP.TM. 8000 spectrophotometer (THERMO
SCIENTIFIC, Wilmington, Del.).
[0239] Samples were tested for insect activity in bioassays
conducted with neonate insect larvae on artificial insect diet. WCR
eggs were obtained from CROP CHARACTERISTICS, INC. (Farmington,
Minn.).
[0240] The bioassays were conducted in 128-well plastic trays
specifically designed for insect bioassays (C-D INTERNATIONAL,
Pitman, N.J.). Each well contained approximately 1.0 mL of an
artificial diet designed for growth of coleopteran insects. A 60
.mu.L aliquot of dsRNA sample was delivered by pipette onto the
surface of the diet of each well (40 .mu.L/cm.sup.2). dsRNA sample
concentrations were calculated as the amount of dsRNA per square
centimeter (ng/cm.sup.2) of surface area (1.5 cm.sup.2) in the
well. The treated trays were held in a fume hood until the liquid
on the diet surface evaporated or was absorbed into the diet.
[0241] Within a few hours of eclosion, individual larvae were
picked up with a moistened camel hair brush and deposited on the
treated diet (one or two larvae per well). The infested wells of
the 128-well plastic trays were then sealed with adhesive sheets of
clear plastic, and vented to allow gas exchange. Bioassay trays
were held under controlled environmental conditions (28.degree. C.,
.about.40% Relative Humidity, 16:8 (Light:Dark)) for 9 days, after
which time the total number of insects exposed to each sample, the
number of dead insects, and the weight of surviving insects were
recorded. Average percent mortality and average growth inhibition
were calculated for each treatment. Growth inhibition (GI) was
calculated as follows:
GI=[1-(TWIT/TNIT)/(TWIBC/TNIBC)] [0242] where TWIT is the Total
Weight of live Insects in the Treatment; [0243] TNIT is the Total
Number of Insects in the Treatment; [0244] TWIBC is the Total
Weight of live Insects in the Background Check (Buffer control);
and [0245] TNIBC is the Total Number of Insects in the Background
Check (Buffer control). Statistical analysis was done using JMP.TM.
software (SAS, Cary, N.C.). LC.sub.50 (Lethal Concentration) is
defined as the dosage at which 50% of the test insects are killed.
GI.sub.50 (Growth Inhibition) is defined as the dosage at which the
mean growth (e.g. live weight) of the test insects is 50% of the
mean value seen in Background Check samples.
[0246] Replicated bioassays demonstrated that ingestion of
particular samples resulted in a surprising and unexpected
mortality and growth inhibition of corn rootworm larvae.
Example 2
Identification of Candidate Target Genes
[0247] Multiple stages of WCR (Diabrotica virgifera virgifera
LeConte) development were selected for pooled transcriptome
analysis to provide candidate target gene sequences for control by
RNAi transgenic plant insect resistance technology.
[0248] In one exemplification, total RNA was isolated from about
0.9 gm whole first-instar WCR larvae; (4 to 5 days post-hatch; held
at 16.degree. C.), and purified using the following phenol/TRI
REAGENT.RTM.-based method (MOLECULAR RESEARCH CENTER, Cincinnati,
Ohio):
[0249] Larvae were homogenized at room temperature in a 15 mL
homogenizer with 10 mL of TRI REAGENT.RTM. until a homogenous
suspension was obtained. Following 5 min. incubation at room
temperature, the homogenate was dispensed into 1.5 mL microfuge
tubes (1 mL per tube), 200 .mu.L of chloroform was added, and the
mixture was vigorously shaken for 15 seconds. After allowing the
extraction to sit at room temperature for 10 min, the phases were
separated by centrifugation at 12,000.times.g at 4.degree. C. The
upper phase (comprising about 0.6 mL) was carefully transferred
into another sterile 1.5 mL tube, and an equal volume of room
temperature isopropanol was added. After incubation at room
temperature for 5 to 10 min, the mixture was centrifuged 8 min at
12,000.times.g (4.degree. C. or 25.degree. C.).
[0250] The supernatant was carefully removed and discarded, and the
RNA pellet was washed twice by vortexing with 75% ethanol, with
recovery by centrifugation for 5 min at 7,500.times.g (4.degree. C.
or 25.degree. C.) after each wash. The ethanol was carefully
removed, the pellet was allowed to air-dry for 3 to 5 min, and then
was dissolved in nuclease-free sterile water. RNA concentration was
determined by measuring the absorbance (A) at 260 nm and 280 nm. A
typical extraction from about 0.9 gm of larvae yielded over 1 mg of
total RNA, with an A.sub.260/A.sub.280 ratio of 1.9. The RNA thus
extracted was stored at -80.degree. C. until further processed.
[0251] RNA quality was determined by running an aliquot through a
1% agarose gel. The agarose gel solution was made using autoclaved
10.times.TAE buffer (Tris-acetate EDTA; 1.times. concentration is
0.04 M Tris-acetate, 1 mM EDTA (ethylenediamine tetra-acetic acid
sodium salt), pH 8.0) diluted with DEPC (diethyl
pyrocarbonate)-treated water in an autoclaved container. 1.times.
TAE was used as the running buffer. Before use, the electrophoresis
tank and the well-forming comb were cleaned with RNaseAway.TM.
(INVITROGEN INC., Carlsbad, Calif.). Two .mu.L of RNA sample were
mixed with 8 .mu.L of TE buffer (10 mM Tris HCl pH 7.0; 1 mM EDTA)
and 10 .mu.L of RNA sample buffer (NOVAGEN.RTM. Catalog No 70606;
EMD4 Bioscience, Gibbstown, N.J.). The sample was heated at
70.degree. C. for 3 min, cooled to room temperature, and 5 .mu.L
(containing 1 .mu.g to 2 .mu.g RNA) were loaded per well.
Commercially available RNA molecular weight markers were
simultaneously run in separate wells for molecular size comparison.
The gel was run at 60 volts for 2 hr.
[0252] A normalized cDNA library was prepared from the larval total
RNA by a commercial service provider (EUROFINS MWG Operon,
Huntsville, Ala.), using random priming. The normalized larval cDNA
library was sequenced at 1/2 plate scale by GS FLX 454 Titanium.TM.
series chemistry at EUROFINS MWG Operon, which resulted in over
600,000 reads with an average read length of 348 bp. 350,000 reads
were assembled into over 50,000 contigs. Both the unassembled reads
and the contigs were converted into BLASTable databases using the
publicly available program, FORMATDB (available from NCBI).
[0253] Total RNA and normalized cDNA libraries were similarly
prepared from materials harvested at other WCR developmental
stages. A pooled transcriptome library for target gene screening
was constructed by combining cDNA library members representing the
various developmental stages.
[0254] Candidate genes for RNAi targeting were selected using
information regarding lethal RNAi effects of particular genes in
other insects such as Drosophila and Tribolium. These genes were
hypothesized to be essential for survival and growth in coleopteran
insects. Selected target gene homologs were identified in the
transcriptome sequence database as described below. Full-length or
partial sequences of the target genes were amplified by PCR to
prepare templates for double-stranded RNA (dsRNA) production.
[0255] TBLASTN searches using candidate protein coding sequences
were run against BLASTable databases containing the unassembled
Diabrotica sequence reads or the assembled contigs. Significant
hits to a Diabrotica sequence (defined as better than e.sup.-20 for
contigs homologies and better than e.sup.-10 for unassembled
sequence reads homologies) were confirmed using BLASTX against the
NCBI non-redundant database. The results of this BLASTX search
confirmed that the Diabrotica homolog candidate gene sequences
identified in the TBLASTN search indeed comprised Diabrotica genes,
or were the best hit to the non-Diabrotica candidate gene sequence
present in the Diabrotica sequences. In most cases, Tribolium
candidate genes which were annotated as encoding a protein gave an
unambiguous sequence homology to a sequence or sequences in the
Diabrotica transcriptome sequences. In a few cases, it was clear
that some of the Diabrotica contigs or unassembled sequence reads
selected by homology to a non-Diabrotica candidate gene overlapped,
and that the assembly of the contigs had failed to join these
overlaps. In those cases, Sequencher.TM. v4.9 (GENE CODES
CORPORATION, Ann Arbor, Mich.) was used to assemble the sequences
into longer contigs.
[0256] A candidate target gene encoding Diabrotica COPI beta (SEQ
ID NO:1) was identified as a gene that may lead to coleopteran pest
mortality, inhibition of growth, inhibition of development, or
inhibition of reproduction in WCR.
[0257] Genes with Homology to WCR COPI Beta
[0258] COPI refers to the specific coat protein complex that
inhibits the budding process on the cis-Golgi membrane (Nickel, et
al. 2002. Journal of Cell Science 115, 3235-3240). The COPI
coatomer complex consists of seven subunits. COPI coatomer BETA is
one of the subunits. The function of the complex is to transport
vesicles from the cis-end of the Golgi complex back to the rough
endoplasmic reticulum, where they were originally synthesized.
Other Diabrotica virgifera proteins that also contain this domain
may share structural and/or functional properties, and thus a gene
that encodes one of these proteins may comprise a candidate target
gene that may lead to coleopteran pest mortality, inhibition of
growth, inhibition of development, or inhibition of reproduction in
WCR.
[0259] The sequence of SEQ ID NO:1 is novel. The sequence is not
provided in public databases and is not disclosed in
WO/2011/025860; U.S. Patent Application No. 20070124836; U.S.
Patent Application No. 20090306189; U.S. Patent Application No.
US20070050860; U.S. Patent Application No. 20100192265;or U.S. Pat.
No. 7,612,194. There was no significant homologous nucleotide
sequence found within a GENBANK search. The closest homolog of the
Diabrotica COPI beta amino acid sequence (SEQ ID NO:2) is a
Tribolium casetanum protein having GENBANK Accession No.
XP_001816488.1 (92% similar; 83% identical over the homology
region).
[0260] COPI beta dsRNA transgenes can be combined with other dsRNA
molecules to provide redundant RNAi targeting and synergistic RNAi
effects. Transgenic corn events expressing dsRNA that targets COPI
beta are useful for preventing root feeding damage by corn
rootworm. COPI beta dsRNA transgenes represent new modes of action
for combining with Bacillus thuringiensis, Alcaligenes spp., or
Pseudomonas spp. insecticidal protein technology in Insect
Resistance Management gene pyramids to mitigate against the
development of rootworm populations resistant to either of these
rootworm control technologies.
[0261] Full-length or partial clones of sequences of a Diabrotica
candidate gene, herein referred to as COPI beta, were used to
generate PCR amplicons for dsRNA synthesis.
[0262] SEQ ID NO:1 shows a 3392 bp DNA sequence of Diabrotica COPI
beta.
[0263] SEQ ID NO:3 shows a 667 bp DNA sequence of COPI beta
reg1.
[0264] SEQ ID NO:68 shows a 384 bp DNA sequence of COPI beta
reg2.
[0265] SEQ ID NO:69 shows a 390 bp DNA sequence of COPI beta
reg1.
[0266] SEQ ID NO:70 shows a 122 bp DNA sequence of COPI beta
ver1.
[0267] SEQ ID NO:71 shows a 100 bp DNA sequence of COPI beta
ver2.
[0268] SEQ ID NO:72 shows a 524 bp DNA sequence of COPI beta
var1.
Example 3
Amplification of Target Genes to Produce dsRNA
[0269] Primers were designed to amplify portions of coding regions
of each target gene by PCR. See Table 1. Where appropriate, a T7
phage promoter sequence (TTAATACGACTCACTATAGGGAGA; SEQ ID NO:4) was
incorporated into the 5' ends of the amplified sense or antisense
strands. See Table 1. Total RNA was extracted from WCR, and
first-strand cDNA was used as template for PCR reactions using
opposing primers positioned to amplify all or part of the native
target gene sequence. dsRNA was also amplified from a DNA clone
comprising the coding region for a yellow fluorescent protein (YFP)
(SEQ ID NO:5; Shagin et al. (2004) Mol. Biol. Evol.
21(5):841-50).
TABLE-US-00006 TABLE 1 Primers and Primer Pairs used to amplify
portions of coding regions of exemplary COPI beta target gene and
YFP negative control gene. SEQ ID Gene ID Primer ID NO: Sequence
Pair 1 COPI beta COPI beta- 6 TTAATACGACTCACTATAGGGAGA reg1 FT7
GTTAGTAAGGACTCTACATTCGTG TTCC COPI beta- 7 TTAATACGACTCACTATAGGGAGA
RT7 GGTTATTGATTTTGTGGGAAGTCC Pair 2 COPI beta COPI beta- 73
TTAATACGACTCACTATAGGGAGA reg2 475-384_F GATGTCGCTGTCACAAGCTTTAGT
COPI beta- 74 TTAATACGACTCACTATAGGGAGA 475-384_R
ACAACATTCCTATCTGACCCCG Pair 3 COPI beta COPI beta- 75
TTAATACGACTCACTATAGGGAGA reg3 475-390_F GGACTATATAGTGCCTGCTAGTTG
TACA COPI beta- 76 TTAATACGACTCACTATAGGGAGA 475-390_R
GCAGCTACTACTTTATGTTGTGTG CT Pair 4 COPI beta COPI beta 77
TTAATACGACTCACTATAGGGAGA ver1 v1_F GAAGAGAATGCTCCTGCACC COPI beta
78 TTAATACGACTCACTATAGGGAGA v1_R AGAGGAGGTCGTGCTTCTTTAG Pair 5 COPI
beta COPI beta 79 TTAATACGACTCACTATAGGGAGA ver2 v2_F
ATTTTGGGAGAATACGCGAG COPI beta 80 TTAATACGACTCACTATAGGGAGA v2_R
TCTGCTCAGCTTCAACGAG Pair 6 COPI beta COPI beta- 81
TTAATACGACTCACTATAGGGAGA var1 150-524_F CCATCGCTGATTACCTCA COPI
beta- 82 TTAATACGACTCACTATAGGGAGA 150-524_R ACATAGTGCGCACGCACTCA
Pair 7 YFP YFP-F_T7 18 TTAATACGACTCACTATAGGGAGA
CACCATGGGCTCCAGCGGCGCCC YFP-R_T7 21 TTAATACGACTCACTATAGGGAGA
AGATCTTGAAGGCGCTCTTCAGG
Example 4
RNAi Constructs
[0270] Template preparation by PCR and dsRNA synthesis.
[0271] A strategy used to provide specific templates for COPI beta
and YFP dsRNA production is shown in FIG. 1. Template DNAs intended
for use in COPI beta dsRNA synthesis were prepared by PCR using the
primer pairs in Table 1 and (as PCR template) first-strand cDNA
prepared from total RNA isolated from WCR first-instar larvae. For
each selected COPI beta and YFP target gene region, PCR
amplifications introduced a T7 promoter sequence at the 5' ends of
the amplified sense and antisense strands (the YFP segment was
amplified from a DNA clone of the YFP coding region). The PCR
products having a T7 promoter sequence at their 5' ends of both
sense and antisense strands for each region of the target genes
were used for dsRNA synthesis. See FIG. 1. The sequences of the
dsRNA templates amplified with the particular primer pairs were:
SEQ ID NO:3 (COPI beta reg1), COPI beta reg2 (SEQ ID NO:68), COPI
beta reg3 (SEQ ID NO:69), COPI beta ver1 (SEQ ID NO:70), COPI beta
ver2 (SEQ ID NO:71), COPI beta var1 (SEQ ID NO:72) and YFP (SEQ ID
NO:7). Double-stranded RNA for insect bioassay was synthesized and
purified using an AMBION.RTM. MEGASCRIPT.RTM. RNAi kit following
the manufacturer's instructions (INVITROGEN). The concentrations of
dsRNAs were measured using a NANODROP.TM. 8000 spectrophotometer
(THERMO SCIENTIFIC, Wilmington, Del.).
[0272] Construction of Plant Transformation Vectors
[0273] Entry vector (pDAB1177219) harboring a target gene construct
for hairpin formation comprising segments of COPI beta (SEQ ID
NO:1) was assembled using a combination of chemically synthesized
fragments (DNA2.0, Menlo Park, Calif.) and standard molecular
cloning methods. Intramolecular hairpin formation by RNA primary
transcripts was facilitated by arranging (within a single
transcription unit) two copies of a segment of COPI beta target
gene sequence in opposite orientation to one another, the two
segments being separated by an ST-LS1 intron sequence (SEQ ID
NO:10; Vancanneyt et al. (1990) Mol. Gen. Genet. 220(2):245-50).
Thus, the primary mRNA transcript contains the two COPI beta gene
segment sequences as large inverted repeats of one another,
separated by the intron sequence. A copy of a maize ubiquitin 1
promoter (U.S. Pat. No. 5,510,474) was used to drive production of
the primary mRNA hairpin transcript, and a fragment comprising a 3'
untranslated region from a maize peroxidase 5 gene (ZmPer5 3'UTR
v2; U.S. Pat. No. 6,699,984) was used to terminate transcription of
the hairpin-RNA-expressing gene.
[0274] Entry vector pDAB117213 comprises a COPI beta hairpin v2-RNA
construct (SEQ ID NO:8) that comprises a segment of COPI beta (SEQ
ID NO:1)
[0275] Entry vector pDAB117213 described above was used in standard
GATEWAY.RTM. recombination reactions with a typical binary
destination vector (pDAB109805) to produce COPI beta hairpin v2 RNA
expression transformation vectors for Agrobacterium-mediated maize
embryo transformations (pDAB114515 and pDAB115770,
respectively).
[0276] A negative control binary vector, pDAB110853, which
comprises a gene that expresses a YFP hairpin dsRNA, was
constructed by means of standard GATEWAY.RTM. recombination
reactions with a typical binary destination vector (pDAB109805) and
entry vector pDAB101670. Entry Vector pDAB101670 comprises a YFP
hairpin sequence (SEQ ID NO:9) under the expression control of a
maize ubiquitin 1 promoter (as above) and a fragment comprising a
3' untranslated region from a maize peroxidase 5 gene (as
above).
[0277] Binary destination vector pDAB109805 comprises a herbicide
resistance gene (aryloxyalknoate dioxygenase; AAD-1 v3) (U.S. Pat.
No. 7,838,733(B2), and Wright et al. (2010) Proc. Natl. Acad. Sci.
U.S.A. 107:20240-5) under the regulation of a sugarcane bacilliform
badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol.
39:1221-30). A synthetic 5'UTR sequence, comprised of sequences
from a Maize Streak Virus (MSV) coat protein gene 5'UTR and intron
6 from a maize Alcohol Dehydrogenase 1 (ADH1) gene, is positioned
between the 3' end of the SCBV promoter segment and the start codon
of the AAD-1 coding region. A fragment comprising a 3' untranslated
region from a maize lipase gene (ZmLip 3'UTR; U.S. Pat. No.
7,179,902) was used to terminate transcription of the AAD-1
mRNA.
[0278] A further negative control binary vector, pDAB101556, which
comprises a gene that expresses a YFP protein, was constructed by
means of standard GATEWAY.RTM. recombination reactions with a
typical binary destination vector (pDAB9989) and entry vector
pDAB100287. Binary destination vector pDAB9989 comprises a
herbicide resistance gene (aryloxyalknoate dioxygenase; AAD-1 v3)
(as above) under the expression regulation of a maize ubiquitin 1
promoter (as above) and a fragment comprising a 3' untranslated
region from a maize lipase gene (ZmLip 3'UTR; as above). Entry
Vector pDAB100287 comprises a YFP coding region (SEQ ID NO:11)
under the expression control of a maize ubiquitin 1 promoter (as
above) and a fragment comprising a 3' untranslated region from a
maize peroxidase 5 gene (as above).
[0279] SEQ ID NO:8 presents an COPI beta hairpin v2-RNA-forming
sequence as found in pDAB117219.
Example 5
Screening of Candidate Target Genes
[0280] Synthetic dsRNA designed to inhibit target gene sequences
identified in EXAMPLE 2 caused mortality and growth inhibition when
administered to WCR in diet-based assays. COPI beta reg1, COPI beta
reg2, COPI beta reg3, COPI beta ver1, COPI beta ver2, COPI beta
var1 were observed to exhibit greatly increased efficacy in this
assay over other dsRNAs screened.
[0281] Replicated bioassays demonstrated that ingestion of dsRNA
preparations derived from COPI beta reg1, COPI beta reg2, COPI beta
reg3, COPI beta ver1, COPI beta ver2, COPI beta var1 resulted in
mortality and/or growth inhibition of western corn rootworm larvae.
Table 2 and Table 3 show the results of diet-based feeding
bioassays of WCR larvae following 9-day exposure to these dsRNAs,
as well as the results obtained with a negative control sample of
dsRNA prepared from a yellow fluorescent protein (YFP) coding
region (SEQ ID NO:5).
TABLE-US-00007 TABLE 2 Results of COPI beta dsRNA diet feeding
assays obtained with western corn rootworm larvae after 9 days of
feeding. ANOVA analysis found significance differences in Mean %
Mortality and Mean % Growth Inhibition (GI). Means were separated
using the Tukey-Kramer test. DOSE MEAN (% MORTALITY) .+-. MEAN (GI)
.+-. GENE NAME (NG/CM.sup.2) NO. ROWS SEM* SEM COPI beta reg1 500 6
63.27 .+-. 7.55 (A) 0.80 .+-. 0.03 (A) COPI beta reg2 500 4 52.94
.+-. 8.66 (AB) 0.75 .+-. 0.04 (A) COPI beta reg3 500 4 49.63 .+-.
8.79 (AB) 0.76 .+-. 0.06 (A) COPI beta ver1 500 10 38.97 .+-. 7.21
(B) 0.77 .+-. 0.09 (A) COPI beta ver2 500 12 48.04 .+-. 4.15 (AB)
0.83 .+-. 0.03 (A) COPI beta var1 500 4 30.66 .+-. 6.33 (BC) 0.40
.+-. 0.22 (AB) TE** 0 20 12.04 .+-. 1.67 (C) -0.01 .+-. 0.01 (B)
WATER 0 20 9.09 .+-. 1.70 (C) -0.07 .+-. 0.06 (B) YFP*** 500 20
8.92 .+-. 1.95 (C) -0.19 .+-. 0.15 (B) *SEM = Standard Error of the
Mean. Letters in parentheses designate statistical levels. Levels
not connected by same letter are significantly different (P <
0.05). **TE = Tris HCl (1 mM) plus EDTA (1 mM) buffer, pH7.2.
***YFP = Yellow Fluorescent Protein
TABLE-US-00008 TABLE 3 Summary of oral potency of COPI beta dsRNA
on WCR larvae (ng/cm.sup.2). LC.sub.50 GI.sub.50 Gene Name
(ng/cm.sup.2) Range (ng/cm.sup.2) Range COPI beta reg1 5.57
0.87-25.52 0.59 0.27-1.26 COPI beta ver2 189.08 519.42-2906.51 0.21
0.03-1.41
[0282] It has previously been suggested that certain genes of
Diabrotica spp. may be exploited for RNAi-mediated insect control.
See U.S. Patent Publication No. 2007/0124836, which discloses 906
sequences, and U.S. Pat. No. 7,612,194, which discloses 9,112
sequences. However, it was determined that many genes suggested to
have utility for RNAi-mediated insect control are not efficacious
in controlling Diabrotica. It was also determined that sequences
COPI beta reg1, COPI beta reg2, COPI beta reg3, COPI beta ver1,
COPI beta ver2, COPI beta var1 provide surprising and unexpected
superior control of Diabrotica, compared to other genes suggested
to have utility for RNAi-mediated insect control.
[0283] For example, Annexin, Beta spectrin 2, and mtRP-L4 were each
suggested in U.S. Pat. No. 7,612,194 to be efficacious in
RNAi-mediated insect control. SEQ ID NO:12 is the DNA sequence of
Annexin region 1 (Reg 1), and SEQ ID NO:13 is the DNA sequence of
Annexin region 2 (Reg 2). SEQ ID NO:14 is the DNA sequence of Beta
spectrin 2 region 1 (Reg 1), and SEQ ID NO:15 is the DNA sequence
of Beta spectrin 2 region 2 (Reg2). SEQ ID NO:16 is the DNA
sequence of mtRP-L4 region 1 (Reg 1), and SEQ ID NO:17 is the DNA
sequence of mtRP-L4 region 2 (Reg 2). A YFP sequence (SEQ ID NO:5)
was also used to produce dsRNA as a negative control.
[0284] Each of the aforementioned sequences was used to produce
dsRNA by the methods of EXAMPLE 3. The strategy used to provide
specific templates for dsRNA production is shown in FIG. 2.
Template DNAs intended for use in dsRNA synthesis were prepared by
PCR using the primer pairs in Table 4 and (as PCR template)
first-strand cDNA prepared from total RNA isolated from WCR
first-instar larvae. (YFP was amplified from a DNA clone.) For each
selected target gene region, two separate PCR amplifications were
performed. The first PCR amplification introduced a T7 promoter
sequence at the 5' end of the amplified sense strands. The second
reaction incorporated the T7 promoter sequence at the 5' ends of
the antisense strands. The two PCR amplified fragments for each
region of the target genes were then mixed in approximately equal
amounts, and the mixture was used as transcription template for
dsRNA production. See FIG. 2. Double-stranded RNA was synthesized
and purified using an AMBION.RTM. MEGAscript.RTM. RNAi kit
following the manufacturer's instructions (INVITROGEN). The
concentrations of dsRNAs were measured using a NANODROP.TM. 8000
spectrophotometer (THERMO SCIENTIFIC, Wilmington, Del.). and the
dsRNAs were each tested by the same diet-based bioassay methods
described above. Table 4 lists the sequences of the primers used to
produce the YFP, Annexin Reg1, Annexin Reg2, Beta spectrin 2 Reg1,
Beta spectrin 2 Reg2, mtRP-L4 Reg1, and mtRP-L4 Reg2 dsRNA
molecules. YFP primer sequences for use in the method depicted in
FIG. 2 are also listed in Table 4. Table 5 presents the results of
diet-based feeding bioassays of WCR larvae following 9-day exposure
to these dsRNA molecules. Replicated bioassays demonstrated that
ingestion of these dsRNAs resulted in no mortality or growth
inhibition of western corn rootworm larvae above that seen with
control samples of TE buffer, Water, or YFP protein.
TABLE-US-00009 TABLE 4 Primers and Primer Pairs used to amplify
portions of coding regions of genes. Gene SEQ ID (Region) Primer ID
NO: Sequence Pair 8 YFP YFP-F_T7 18 TTAATACGACTCACTATAGGGAGAC
ACCATGGGCTCCAGCGGCGCCC YFP-R 19 AGATCTTGAAGGCGCTCTTCAGG Pair 9 YFP
YFP-F 20 CACCATGGGCTCCAGCGGCGCCC YFP-R_T7 21
TTAATACGACTCACTATAGGGAGAA GATCTTGAAGGCGCTCTTCAGG Pair 10 Annexin
Ann-F1_T7 22 TTAATACGACTCACTATAGGGAGAG (Reg 1)
CTCCAACAGTGGTTCCTTATC Annexin Ann-R1 23 CTAATAATTCTTTTTTAATGTTCCTG
(Reg 1) AGG Pair 11 Annexin Ann-F1 24 GCTCCAACAGTGGTTCCTTATC (Reg
1) Annexin Ann-R1_T7 25 TTAATACGACTCACTATAGGGAGAC (Reg 1)
TAATAATTCTTTTTTAATGTTCCTGA GG Pair 12 Annexin Ann-F2_T7 26
TTAATACGACTCACTATAGGGAGAT (Reg 2) TGTTACAAGCTGGAGAACTTCTC Annexin
Ann-R2 27 CTTAACCAACAACGGCTAATAAGG (Reg 2) Pair 13 Annexin Ann-F2
28 TTGTTACAAGCTGGAGAACTTCTC (Reg 2) Annexin Ann-R2T7 29
TTAATACGACTCACTATAGGGAGAC (Reg 2) TTAACCAACAACGGCTAATAAGG Pair 14
Beta-spect2 Betasp2-F1_T7 30 TTAATACGACTCACTATAGGGAGAA (Reg 1)
GATGTTGGCTGCATCTAGAGAA Beta-spect2 Betasp2-R1 31
GTCCATTCGTCCATCCACTGCA (Reg 1) Pair 15 Beta-spect2 Betasp2-F1 32
AGATGTTGGCTGCATCTAGAGAA (Reg 1) Beta-spect2 Betasp2-R1_T7 33
TTAATACGACTCACTATAGGGAGAG (Reg 1) TCCATTCGTCCATCCACTGCA Pair 16
Beta-spect2 Betasp2-F2_T7 34 TTAATACGACTCACTATAGGGAGAG (Reg 2)
CAGATGAACACCAGCGAGAAA Beta-spect2 Betasp2-R2 35
CTGGGCAGCTTCTTGTTTCCTC (Reg 2) Pair 17 Beta-spect2 Betasp2-F2 36
GCAGATGAACACCAGCGAGAAA (Reg 2) Beta-spect2 Betasp2-R2_T7 37
TTAATACGACTCACTATAGGGAGAC (Reg 2) TGGGCAGCTTCTTGTTTCCTC Pair 18
mtRP-L4 L4-F1_T7 38 TTAATACGACTCACTATAGGGAGAA (Reg 1)
GTGAAATGTTAGCAAATATAACATCC mtRP-L4 L4-R1 39
ACCTCTCACTTCAAATCTTGACTTTG (Reg 1) Pair 19 mtRP-L4 L4-F1 40
AGTGAAATGTTAGCAAATATAACAT (Reg 1) CC mtRP-L4 L4-R1_T7 41
TTAATACGACTCACTATAGGGAGAA (Reg 1) CCTCTCACTTCAAATCTTGACTTTG Pair 20
mtRP-L4 L4-F2_T7 42 TTAATACGACTCACTATAGGGAGAC (Reg 2)
AAAGTCAAGATTTGAAGTGAGAGGT mtRP-L4 L4-R2 43
CTACAAATAAAACAAGAAGGACCCC (Reg 2) Pair 21 mtRP-L4 L4-F2 44
CAAAGTCAAGATTTGAAGTGAGAGGT (Reg 2) mtRP-L4 L4-R2_T7 45
TTAATACGACTCACTATAGGGAGAC (Reg 2) TACAAATAAAACAAGAAGGACCCC
TABLE-US-00010 TABLE 5 Results of diet feeding assays obtained with
western corn rootworm larvae after 9 days. Mean Live Mean Dose
Larval Mean % Growth Gene Name (ng/cm.sup.2) Weight (mg) Mortality
Inhibition Annexin-Reg 1 1000 0.545 0 -0.262 Annexin-Reg 2 1000
0.565 0 -0.301 Beta spectrin2 Reg 1 1000 0.340 12 -0.014 Beta
spectrin2 Reg 2 1000 0.465 18 -0.367 mtRP-L4 Reg 1 1000 0.305 4
-0.168 mtRP-L4 Reg 2 1000 0.305 7 -0.180 TE buffer* 0 0.430 13
0.000 Water 0 0.535 12 0.000 YFP** 1000 0.480 9 -0.386 *TE = Tris
HCl (10 mM) plus EDTA (1 mM) buffer, pH 8. **YFP = Yellow
Fluorescent Protein
Example 6
Production of Transgenic Maize Tissues Comprising Insecticidal
Hairpin dsRNAs
[0285] Agrobacterium-Mediated Transformation
[0286] Transgenic maize cells, tissues, and plants that produce one
or more insecticidal dsRNA molecules (for example, at least one
dsRNA molecule including a dsRNA molecule targeting a gene
comprising COPI beta; SEQ ID NO:1) through expression of a chimeric
gene stably-integrated into the plant genome were produced
following Agrobacterium-mediated transformation. Maize
transformation methods employing superbinary or binary
transformation vectors are known in the art, as described, for
example, in U.S. Pat. No. 8,304,604, which is herein incorporated
by reference in its entirety. Transformed tissues were selected by
their ability to grow on Haloxyfop-containing medium and were
screened for dsRNA production, as appropriate. Portions of such
transformed tissue cultures may be presented to neonate corn
rootworm larvae for bioassay, essentially as described in EXAMPLE
1.
[0287] Agrobacterium Culture Initiation
[0288] Glycerol stocks of Agrobacterium strain DAt13192 cells (WO
2012/016222A2) harboring a binary transformation vector pDAB114515,
pDAB115770, pDAB110853 or pDAB101556 described above (EXAMPLE 4)
were streaked on AB minimal medium plates (Watson, et al., (1975)
J. Bacteriol. 123:255-264) containing appropriate antibiotics and
were grown at 20.degree. C. for 3 days. The cultures were then
streaked onto YEP plates (gm/L: yeast extract, 10; Peptone, 10;
NaCl 5) containing the same antibiotics and were incubated at
20.degree. C. for 1 day.
[0289] Agrobacterium Culture
[0290] On the day of an experiment, a stock solution of Inoculation
Medium and acetosyringone was prepared in a volume appropriate to
the number of constructs in the experiment and pipetted into a
sterile, disposable, 250 mL flask. Inoculation Medium (Frame et al.
(2011) Genetic Transformation Using Maize Immature Zygotic Embryos.
IN Plant Embryo Culture Methods and Protocols: Methods in Molecular
Biology. T. A. Thorpe and E. C. Yeung, (Eds), Springer Science and
Business Media, LLC. pp 327-341) contained: 2.2 gm/L MS salts;
1.times.ISU Modified MS Vitamins (Frame et al., ibid.) 68.4 gm/L
sucrose; 36 gm/L glucose; 115 mg/L L-proline; and 100 mg/L
myo-inositol; at pH 5.4.) Acetosyringone was added to the flask
containing Inoculation Medium to a final concentration of 200 .mu.M
from a 1 M stock solution in 100% dimethyl sulfoxide and the
solution was thoroughly mixed.
[0291] For each construct, 1 or 2 inoculating loops-full of
Agrobacterium from the YEP plate were suspended in 15 mL of the
Inoculation Medium/acetosyringone stock solution in a sterile,
disposable, 50 mL centrifuge tube, and the optical density of the
solution at 550 nm (OD550) was measured in a spectrophotometer. The
suspension was then diluted to OD550 of 0.3 to 0.4 using additional
Inoculation Medium/acetosyringone mixture. The tube of
Agrobacterium suspension was then placed horizontally on a platform
shaker set at about 75 rpm at room temperature and shaken for 1 to
4 hours while embryo dissection was performed.
[0292] Ear Sterilization and Embryo Isolation
[0293] Maize immature embryos were obtained from plants of Zea mays
inbred line B104 (Hallauer et al. (1997) Crop Science 37:1405-1406)
grown in the greenhouse and self- or sib-pollinated to produce
ears. The ears were harvested approximately 10 to 12 days
post-pollination. On the experimental day, de-husked ears were
surface-sterilized by immersion in a 20% solution of commercial
bleach (ULTRA CLOROX.RTM. Germicidal Bleach, 6.15% sodium
hypochlorite; with two drops of TWEEN 20) and shaken for 20 to 30
min, followed by three rinses in sterile deionized water in a
laminar flow hood. Immature zygotic embryos (1.8 to 2.2 mm long)
were aseptically dissected from each ear and randomly distributed
into microcentrifuge tubes containing 2.0 mL of a suspension of
appropriate Agrobacterium cells in liquid Inoculation Medium with
200 .mu.M acetosyringone, into which 2 .mu.L of 10% BREAK-THRU.RTM.
5233 surfactant (EVONIK INDUSTRIES; Essen, Germany) had been added.
For a given set of experiments, embryos from pooled ears were used
for each transformation.
[0294] Agrobacterium Co-Cultivation
[0295] Following isolation, the embryos were placed on a rocker
platform for 5 minutes. The contents of the tube were then poured
onto a plate of Co-cultivation Medium, which contained 4.33 gm/L MS
salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L
L-proline; 3.3 mg/L Dicamba in KOH (3,6-dichloro-o-anisic acid or
3,6-dichloro-2-methoxybenzoic acid); 100 mg/L myo-inositol; 100
mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 200 .mu.M
acetosyringone in DMSO; and 3 gm/L GELZAN.TM., at pH 5.8. The
liquid Agrobacterium suspension was removed with a sterile,
disposable, transfer pipette. The embryos were then oriented with
the scutellum facing up using sterile forceps with the aid of a
microscope. The plate was closed, sealed with 3M.TM. MICROPORE.TM.
medical tape, and placed in an incubator at 25.degree. C. with
continuous light at approximately 60 .mu.mol m.sup.-2s.sup.-1 of
Photosynthetically Active Radiation (PAR).
[0296] Callus Selection and Regeneration of Transgenic Events
[0297] Following the Co-Cultivation period, embryos were
transferred to Resting Medium, which was composed of 4.33 gm/L MS
salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L
L-proline; 3.3 mg/L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L
Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 0.5 gm/L MES
(2-(N-morpholino)ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIES
LABR.; Lenexa, Kans.); 250 mg/L Carbenicillin; and 2.3 gm/L
GELZAN.TM.; at pH 5.8. No more than 36 embryos were moved to each
plate. The plates were placed in a clear plastic box and incubated
at 27.degree. C. with continuous light at approximately 50 .mu.mol
m.sup.-2s.sup.-1 PAR for 7 to 10 days. Callused embryos were then
transferred (<18/plate) onto Selection Medium I, which was
comprised of Resting Medium (above) with 100 nM R-Haloxyfop acid
(0.0362 mg/L; for selection of calli harboring the AAD-1 gene). The
plates were returned to clear boxes and incubated at 27.degree. C.
with continuous light at approximately 50 .mu.mol m.sup.-2s.sup.-1
PAR for 7 days. Callused embryos were then transferred
(<12/plate) to Selection Medium II, which is comprised of
Resting Medium (above) with 500 nM R-Haloxyfop acid (0.181 mg/L).
The plates were returned to clear boxes and incubated at 27.degree.
C. with continuous light at approximately 50 .mu.mol
m.sup.-2s.sup.-1 PAR for 14 days. This selection step allowed
transgenic callus to further proliferate and differentiate.
[0298] Proliferating, embryogenic calli were transferred
(<9/plate) to Pre-Regeneration medium. Pre-Regeneration Medium
contained 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 45
gm/L sucrose; 350 mg/L L-proline; 100 mg/L myo-inositol; 50 mg/L
Casein Enzymatic Hydrolysate; 1.0 mg/L AgNO.sub.3; 0.25 gm/L MES;
0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid in
ethanol; 1 mg/L 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5
gm/L GELZAN.TM.; and 0.181 mg/L Haloxyfop acid; at pH 5.8. The
plates were stored in clear boxes and incubated at 27.degree. C.
with continuous light at approximately 50 .mu.mol m.sup.-2s.sup.-1
PAR for 7 days. Regenerating calli were then transferred
(<6/plate) to Regeneration Medium in PHYTATRAYS.TM.
(SIGMA-ALDRICH) and incubated at 28.degree. C. with 16 hours
light/8 hours dark per day (at approximately 160 .mu.mol
m.sup.-2s.sup.-1 PAR) for 14 days or until shoots and roots
developed. Regeneration Medium contained 4.33 gm/L MS salts;
1.times.ISU Modified MS Vitamins; 60 gm/L sucrose; 100 mg/L
myo-inositol; 125 mg/L Carbenicillin; 3 gm/L GELLAN.TM. gum; and
0.181 mg/L R-Haloxyfop acid; at pH 5.8. Small shoots with primary
roots were then isolated and transferred to Elongation Medium
without selection. Elongation Medium contained 4.33 gm/L MS salts;
1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; and 3.5 gm/L
GELRITE.TM.: at pH 5.8.
[0299] Transformed plant shoots selected by their ability to grow
on medium containing Haloxyfop were transplanted from
PHYTATRAYS.TM. to small pots filled with growing medium (PROMIX BX;
PREMIER TECH HORTICULTURE), covered with cups or HUMI-DOMES (ARCO
PLASTICS), and then hardened-off in a CONVIRON growth chamber
(27.degree. C. day/24.degree. C. night, 16-hour photoperiod, 50-70%
RH, 200 .mu.mol m.sup.-2s.sup.-1 PAR). In some instances, putative
transgenic plantlets were analyzed for transgene relative copy
number by quantitative real-time PCR assays using primers designed
to detect the AAD1 herbicide tolerance gene integrated into the
maize genome. Further, RNA qPCR assays were used to detect the
presence of the ST-LS1 intron sequence in expressed dsRNAs of
putative transformants. Selected transformed plantlets were then
moved into a greenhouse for further growth and testing.
[0300] Transfer and Establishment of to Plants in the Greenhouse
for Bioassay and Seed Production
[0301] When plants reached the V3-V4 stage, they were transplanted
into IE CUSTOM BLEND (PROFILE/METRO MIX 160) soil mixture and grown
to flowering in the greenhouse (Light Exposure Type: Photo or
Assimilation; High Light Limit: 1200 PAR; 16-hour day length;
27.degree. C. day/24.degree. C. night).
[0302] Plants to be used for insect bioassays were transplanted
from small pots to TINUS.TM. 350-4 ROOTRAINERS.RTM.
(SPENCER-LEMAIRE INDUSTRIES, Acheson, Alberta, Canada); (one plant
per event per ROOTRAINER.RTM.). Approximately four days after
transplanting to ROOTRAINERS.RTM., plants were infested for
bioassay.
[0303] Plants of the T.sub.1 generation were obtained by
pollinating the silks of T.sub.0 transgenic plants with pollen
collected from plants of non-transgenic elite inbred line B104 or
other appropriate pollen donors, and planting the resultant seeds.
Reciprocal crosses were performed when possible.
Example 7
Molecular Analyses of Transgenic Maize Tissues
[0304] Molecular analyses (e.g. RNA qPCR) of maize tissues were
performed on samples from leaves and roots that were collected from
greenhouse grown plants on the same days that root feeding damage
was assessed.
[0305] Results of RNA qPCR assays for the Per5 3'UTR were used to
validate expression of hairpin transgenes. (A low level of Per5
3'UTR detection is expected in nontransformed maize plants, since
there is usually expression of the endogenous Per5 gene in maize
tissues.) Results of RNA qPCR assays for the ST-LS1 intron sequence
(which is integral to the formation of dsRNA hairpin molecules) in
expressed RNAs were used to validate the presence of hairpin
transcripts. Transgene RNA expression levels were measured relative
to the RNA levels of an endogenous maize gene.
[0306] DNA qPCR analyses to detect a portion of the AAD1 coding
region in genomic DNA were used to estimate transgene insertion
copy number. Samples for these analyses were collected from plants
grown in environmental chambers. Results were compared to DNA qPCR
results of assays designed to detect a portion of a single-copy
native gene, and simple events (having one or two copies of COPI
beta transgenes) were advanced for further studies in the
greenhouse.
[0307] Additionally, qPCR assays designed to detect a portion of
the spectinomycin-resistance gene (SpecR; harbored on the binary
vector plasmids outside of the T-DNA) were used to determine if the
transgenic plants contained extraneous integrated plasmid backbone
sequences.
[0308] Hairpin RNA Transcript Expression Level: Per 5 3'UTR
qPCR
[0309] Callus cell events or transgenic plants were analyzed by
real time quantitative PCR (qPCR) of the Per 5 3'UTR sequence to
determine the relative expression level of the full length hairpin
transcript, as compared to the transcript level of an internal
maize gene (SEQ ID NO:46; GENBANK Accession No. BT069734), which
encodes a TIP41-like protein (i.e. a maize homolog of GENBANK
Accession No. AT4G34270; having a tBLASTX score of 74% identity).
RNA was isolated using an RNAEASY.TM. 96 kit (QIAGEN, Valencia,
Calif.). Following elution, the total RNA was subjected to a DNAse1
treatment according to the kit's suggested protocol. The RNA was
then quantified on a NANODROP 8000 spectrophotometer (THERMO
SCIENTIFIC) and concentration was normalized to 25 ng/.mu.L. First
strand cDNA was prepared using a HIGH CAPACITY cDNA SYNTHESIS KIT
(INVITROGEN) in a 10 .mu.L reaction volume with 5 .mu.L denatured
RNA, substantially according to the manufacturer's recommended
protocol. The protocol was modified slightly to include the
addition of 10 .mu.L of 100 .mu.M T20VN oligonucleotide (IDT) (SEQ
ID NO:47; TTTTTTTTTTTTTTTTTTTTVN, where V is A, C, or G, and N is
A, C, G, or T/U) into the 1 mL tube of random primer stock mix, in
order to prepare a working stock of combined random primers and
oligo dT.
[0310] Following cDNA synthesis, samples were diluted 1:3 with
nuclease-free water, and stored at -20.degree. C. until
assayed.
[0311] Separate real-time PCR assays for the Per5 3' UTR and
TIP41-like transcript were performed on a LIGHTCYCLER.TM. 480
(ROCHE DIAGNOSTICS, Indianapolis, Ind.) in 10 .mu.L reaction
volumes. For the Per5 3'UTR assay, reactions were run with Primers
P5U76S (F) (SEQ ID NO:48) and P5U76A (R) (SEQ ID NO:49), and a
ROCHE UNIVERSAL PROBE.TM. (UPL76; Catalog No. 4889960001; labeled
with FAM). For the TIP41-like reference gene assay, primers TIPmxF
(SEQ ID NO:50) and TIPmxR (SEQ ID NO:51), and Probe HXTIP (SEQ ID
NO:52) labeled with HEX (hexachlorofluorescein) were used.
[0312] All assays included negative controls of no-template (mix
only). For the standard curves, a blank (water in source well) was
also included in the source plate to check for sample
cross-contamination. Primer and probe sequences are set forth in
Table 6. Reaction components recipes for detection of the various
transcripts are disclosed in Table 7, and PCR reactions conditions
are summarized in Table 8. The FAM (6-Carboxy Fluorescein Amidite)
fluorescent moiety was excited at 465 nm and fluorescence was
measured at 510 nm; the corresponding values for the HEX
(hexachlorofluorescein) fluorescent moiety were 533 nm and 580
nm.
TABLE-US-00011 TABLE 6 Oligonucleotide sequences used for molecular
analyses of transcript levels in transgenic maize. SEQ ID Target
Oligonucleotide NO. Sequence Per5 3'UTR P5U76S (F) 48
TTGTGATGTTGGTGGCGTAT Per5 3'UTR P5U76A (R) 49
TGTTAAATAAAACCCCAAAGATCG Per5 3'UTR Roche UPL76 NAv** Roche
Diagnostics Catalog Number 488996001 (FAM-Probe) TIP41 TIPmxF 50
TGAGGGTAATGCCAACTGGTT TIP41 TIPmxR 51 GCAATGTAACCGAGTGTCTCTCAA
TIP41 HXTIP 52 TTTTTGGCTTAGAGTTGATGGTGTACTGA (HEX-Probe) TGA
*TIP41-like protein. **NAv Sequence Not Available from the
supplier.
TABLE-US-00012 TABLE 7 PCR reaction recipes for transcript
detection. Per5 3'UTR TIP-like Gene Component Final Concentration
Roche Buffer 1 X 1X P5U76S (F) 0.4 .mu.M 0 P5U76A (R) 0.4 .mu.M 0
Roche UPL76 (FAM) 0.2 .mu.M 0 HEXtipZM F 0 0.4 .mu.M HEXtipZM R 0
0.4 .mu.M HEXtipZMP (HEX) 0 0.2 .mu.M cDNA (2.0 .mu.L) NA NA Water
To 10 .mu.L To 10 .mu.L
TABLE-US-00013 TABLE 8 Thermocycler conditions for RNA qPCR. Per5
3'UTR and TIP41-like Gene Detection Process Temp. Time No. Cycles
Target Activation 95.degree. C. 10 min 1 Denature 95.degree. C. 10
sec 40 Extend 60.degree. C. 40 sec Acquire FAM or HEX 72.degree. C.
1 sec Cool 40.degree. C. 10 sec 1
[0313] Data were analyzed using LIGHTCYCLER.TM. Software v1.5 by
relative quantification using a second derivative max algorithm for
calculation of Cq values according to the supplier's
recommendations. For expression analyses, expression values were
calculated using the .DELTA..DELTA.Ct method (i.e., 2-(Cq TARGET-Cq
REF)), which relies on the comparison of differences of Cq values
between two targets, with the base value of 2 being selected under
the assumption that, for optimized PCR reactions, the product
doubles every cycle.
[0314] Hairpin Transcript Size and Integrity: Northern Blot
Assay
[0315] In some instances, additional molecular characterization of
the transgenic plants is obtained by the use of Northern Blot (RNA
blot) analysis to determine the molecular size of the COPI beta
hairpin v2 RNA in transgenic plants expressing a COPI beta hairpin
v2 dsRNA.
[0316] All materials and equipment are treated with RNAZAP
(AMBION/INVITROGEN) before use. Tissue samples (100 mg to 500 mg)
are collected in 2 mL SAFELOCK EPPENDORF tubes, disrupted with a
KLECKO.TM. tissue pulverizer (GARCIA MANUFACTURING, Visalia,
Calif.) with three tungsten beads in 1 mL of TRIZOL (INVITROGEN)
for 5 min, then incubated at room temperature (RT) for 10 min.
Optionally, the samples are centrifuged for 10 min at 4.degree. C.
at 11,000 rpm and the supernatant is transferred into a fresh 2 mL
SAFELOCK EPPENDORF tube. After 200 .mu.L of chloroform are added to
the homogenate, the tube is mixed by inversion for 2 to 5 min,
incubated at RT for 10 minutes, and centrifuged at 12,000.times.g
for 15 min at 4.degree. C. The top phase is transferred into a
sterile 1.5 mL EPPENDORF tube, 600 .mu.L of 100% isopropanol are
added, followed by incubation at RT for 10 min to 2 hr, then
centrifuged at 12,000.times.g for 10 min at 4.degree. to 25.degree.
C. The supernatant is discarded and the RNA pellet is washed twice
with 1 mL of 70% ethanol, with centrifugation at 7,500.times.g for
10 min at 4.degree. to 25.degree. C. between washes. The ethanol is
discarded and the pellet is briefly air dried for 3 to 5 min before
resuspending in 50 .mu.L of nuclease-free water.
[0317] Total RNA is quantified using the NANODROP 8000.RTM.
(THERMO-FISHER) and samples are normalized to 5 .mu.g/10 .mu.L. 10
.mu.L of glyoxal (AMBION/INVITROGEN) are then added to each sample.
Five to 14 ng of DIG RNA standard marker mix (ROCHE APPLIED
SCIENCE, Indianapolis, Ind.) are dispensed and added to an equal
volume of glyoxal. Samples and marker RNAs are denatured at
50.degree. C. for 45 min and stored on ice until loading on a 1.25%
SEAKEM GOLD agarose (LONZA, Allendale, N.J.) gel in NORTHERNMAX
10.times. glyoxal running buffer (AMBION/INVITROGEN). RNAs are
separated by electrophoresis at 65 volts/30 mA for 2 hr and 15
min.
[0318] Following electrophoresis, the gel is rinsed in 2.times.SSC
for 5 min and imaged on a GEL DOC station (BIORAD, Hercules,
Calif.), then the RNA is passively transferred to a nylon membrane
(MILLIPORE) overnight at RT, using 10.times.SSC as the transfer
buffer (20.times.SSC consists of 3 M sodium chloride and 300 mM
trisodium citrate, pH 7.0). Following the transfer, the membrane is
rinsed in 2.times.SSC for 5 minutes, the RNA is UV-crosslinked to
the membrane (AGILENT/STRATAGENE), and the membrane is allowed to
dry at RT for up to 2 days.
[0319] The membrane is prehybridized in ULTRAHYB buffer
(AMBION/INVITROGEN) for 1 to 2 hr. The probe consists of a PCR
amplified product containing the sequence of interest, (for
example, the antisense sequence portion of SEQ ID NO:8 as
appropriate) labeled with digoxygenin by means of a ROCHE APPLIED
SCIENCE DIG procedure. Hybridization in recommended buffer is
overnight at a temperature of 60.degree. C. in hybridization tubes.
Following hybridization, the blot is subjected to DIG washes,
wrapped, exposed to film for 1 to 30 minutes, then the film is
developed, all by methods recommended by the supplier of the DIG
kit.
[0320] Transgene Copy Number Determination
[0321] Maize leaf pieces approximately equivalent to 2 leaf punches
were collected in 96-well collection plates (QIAGEN). Tissue
disruption was performed with a KLECKO.TM. tissue pulverizer
(GARCIA MANUFACTURING, Visalia, Calif.) in BIOSPRINT96 AP1 lysis
buffer (supplied with a BIOSPRINT96 PLANT KIT; QIAGEN) with one
stainless steel bead. Following tissue maceration, genomic DNA
(gDNA) was isolated in high throughput format using a BIOSPRINT96
PLANT KIT and a BIOSPRINT96 extraction robot. Genomic DNA was
diluted 2:3 DNA:water prior to setting up the qPCR reaction.
[0322] qPCR Analysis
[0323] Transgene detection by hydrolysis probe assay was performed
by real-time PCR using a LIGHTCYCLER.RTM.480 system.
Oligonucleotides to be used in hydrolysis probe assays to detect
the ST-LS1 intron sequence (SEQ ID NO:10), or to detect a portion
of the SpecR gene (i.e. the spectinomycin resistance gene borne on
the binary vector plasmids; SEQ ID NO:64; SPC1 oligonucleotides in
Table 9), were designed using LIGHTCYCLER.RTM. PROBE DESIGN
SOFTWARE 2.0. Further, oligonucleotides to be used in hydrolysis
probe assays to detect a segment of the AAD-1 herbicide tolerance
gene (SEQ ID NO:58; GAAD1 oligonucleotides in Table 9) were
designed using PRIMER EXPRESS software (APPLIED BIOSYSTEMS). Table
9 shows the sequences of the primers and probes. Assays were
multiplexed with reagents for an endogenous maize chromosomal gene
(Invertase (SEQ ID NO:55; GENBANK Accession No: U16123; referred to
herein as IVR1), which served as an internal reference sequence to
ensure gDNA was present in each assay. For amplification,
LIGHTCYCLER.RTM.480 PROBES MASTER mix (ROCHE APPLIED SCIENCE) was
prepared at 1.times. final concentration in a 10 .mu.L volume
multiplex reaction containing 0.4 .mu.M of each primer and 0.2
.mu.M of each probe (Table 10). A two step amplification reaction
was performed as outlined in Table 11. Fluorophore activation and
emission for the FAM- and HEX-labeled probes were as described
above; CY5 conjugates are excited maximally at 650 nm and fluoresce
maximally at 670 nm.
[0324] Cp scores (the point at which the fluorescence signal
crosses the background threshold) were determined from the real
time PCR data using the fit points algorithm (LIGHTCYCLER.RTM.
SOFTWARE release 1.5) and the Relative Quant module (based on the
.DELTA..DELTA.Ct method). Data were handled as described previously
(above; RNA qPCR).
TABLE-US-00014 TABLE 9 Sequences of primers and probes (with
fluorescent conjugate) used for gene copy number determinations and
binary vector plasmid backbone detection. SEQ ID Name NO: Sequence
ST-LS1-F 65 GTATGTTTCTGCTTCTACCTTTGAT ST-LS1-R 66
CCATGTTTTGGTCATATATTAGAAAAGTT ST-LS1-P 67
AGTAATATAGTATTTCAAGTATTTTTTTCAAAAT (FAM) GAAD1-F 56
TGTTCGGTTCCCTCTACCAA GAAD1-R 57 CAACATCCATCACCTTGACTGA GAAD1-P
(FAM) 58 CACAGAACCGTCGCTTCAGCAACA IVR1-F 59 TGGCGGACGACGACTTGT
IVR1-R 60 AAAGTTTGGAGGCTGCCGT IVR1-P (HEX) 61
CGAGCAGACCGCCGTGTACTTCTACC SPC1A 62 CTTAGCTGGATAACGCCAC SPC1S 63
GACCGTAAGGCTTGATGAA TQSPEC (CY5*) 64 CGAGATTCTCCGCGCTGTAGA CY5 =
Cyanine-5
TABLE-US-00015 TABLE 10 Reaction components for gene copy number
analyses and plasmid backbone detection. Component Amt. (.mu.L)
Stock Final Conc'n 2x Buffer 5.0 2x 1x Appropriate Forward Primer
0.4 10 .mu.M 0.4 Appropriate Reverse Primer 0.4 10 .mu.M 0.4
Appropriate Probe 0.4 5 .mu.M 0.2 IVR1-Forward Primer 0.4 10 .mu.M
0.4 IVR1-Reverse Primer 0.4 10 .mu.M 0.4 IVR1-Probe 0.4 5 .mu.M 0.2
H.sub.2O 0.6 NA* NA gDNA 2.0 ND** ND Total 10.0 *NA = Not
Applicable **ND = Not Determined
TABLE-US-00016 TABLE 11 Thermocycler conditions for DNA qPCR
Genomic copy number analyses Process Temp. Time No. Cycles Target
Activation 95.degree. C. 10 min 1 Denature 95.degree. C. 10 sec 40
Extend & Acquire 60.degree. C. 40 sec FAM, HEX, or CY5 Cool
40.degree. C. 10 sec 1
Example 8
Bioassay of Transgenic Maize
[0325] Insect Bioassays
[0326] Bioactivity of dsRNA of the subject invention produced in
plant cells is demonstrated by bioassay methods. See, e.g., Baum et
al. (2007) Nat. Biotechnol. 25(11):1322-1326. One is able to
demonstrate efficacy, for example, by feeding various plant tissues
or tissue pieces derived from a plant producing an insecticidal
dsRNA to target insects in a controlled feeding environment.
Alternatively, extracts are prepared from various plant tissues
derived from a plant producing the insecticidal dsRNA and the
extracted nucleic acids are dispensed on top of artificial diets
for bioassays as previously described herein. The results of such
feeding assays are compared to similarly conducted bioassays that
employ appropriate control tissues from host plants that do not
produce an insecticidal dsRNA, or to other control samples.
[0327] Insect Bioassays with Transgenic Maize Events.
[0328] Two western corn rootworm larvae (1 to 3 days old) hatched
from washed eggs are selected and placed into each well of the
bioassay tray. The wells are then covered with a "PULL N' PEEL" tab
cover (BIO-CV-16, BIO-SERV) and placed in a 28.degree. C. incubator
with an 18 hr/6 hr light/dark cycle. Nine days after the initial
infestation, the larvae are assessed for mortality, which is
calculated as the percentage of dead insects out of the total
number of insects in each treatment. The insect samples are frozen
at -20.degree. C. for two days, then the insect larvae from each
treatment are pooled and weighed. The percent of growth inhibition
is calculated as the mean weight of the experimental treatments
divided by the mean of the average weight of two control well
treatments. The data are expressed as a Percent Growth Inhibition
(of the Negative Controls). Mean weights that exceed the control
mean weight are normalized to zero.
[0329] Insect Bioassays in the Greenhouse.
[0330] Western corn rootworm (WCR, Diabrotica virgifera virgifera
LeConte) eggs were received in soil from CROP CHARACTERISTICS
(Farmington, Minn.). WCR eggs were incubated at 28.degree. C. for
10 to 11 days. Eggs were washed from the soil, placed into a 0.15%
agar solution, and the concentration was adjusted to approximately
75 to 100 eggs per 0.25 mL aliquot. A hatch plate was set up in a
Petri dish with an aliquot of egg suspension to monitor hatch
rates.
[0331] The soil around the maize plants growing in ROOTRAINERS.RTM.
was infested with 150 to 200 WCR eggs. The insects were allowed to
feed for 2 weeks, after which time a "Root Rating" was given to
each plant. A Node-Injury Scale was utilized for grading
essentially according to Oleson et al. (2005, J. Econ. Entomol.
98:1-8). Plants which passed this bioassay were transplanted to
5-gallon pots for seed production. Transplants were treated with
insecticide to prevent further rootworm damage and insect release
in the greenhouses. Plants were hand pollinated for seed
production. Seeds produced by these plants were saved for
evaluation at the T.sub.1 and subsequent generations of plants.
[0332] Greenhouse bioassays included two kinds of negative control
plants. Transgenic negative control plants were generated by
transformation with vectors harboring genes designed to produce a
yellow fluorescent protein (YFP) or a YFP hairpin dsRNA (See
Example 4). Nontransformed negative control plants were grown from
seeds of lines 7sh382 or B104. Bioassays were conducted on two
separate dates, with negative controls included in each set of
plant materials.
[0333] Table 12 shows the combined results of molecular analyses
and bioassays for COPI beta-hairpin plants. Examination of the
bioassay results summarized in Table 12 reveals the surprising and
unexpected observation that the majority of the transgenic maize
plants harboring constructs that express an COPI beta hairpin v2
dsRNA comprising segments of SEQ ID NO:1, for example, as
exemplified in SEQ ID NO:8, are protected against root damage
incurred by feeding of western corn rootworm larvae. Twenty-two of
the 37 graded events had a root rating of 0.5 or lower. Table 13
shows the combined results of molecular analyses and bioassays for
negative control plants. Most of the plants had no protection
against WCR larvae feeding, although five of the 34 graded plants
had a root rating of 0.75 or lower. The presence of some plants
having low root ratings scores amongst the negative control plant
set is sometimes observed and reflects the variability and
difficulty of conducting this type of bioassay in a greenhouse
setting.
TABLE-US-00017 TABLE 12 Greenhouse bioassay and molecular analyses
results of COPI beta hairpin v2-expressing maize plants. Leaf
Tissue Root Tissue PER5 PER5 Batch ST-LS1 UTR ST-LS1 UTR Root
Sample ID # RTL* RTL RTL* RTL Rating COPI beta v2 Hairpin Events
117219[1]-001.001 1 0.633 398.9 2.928 729.1 0.1 117219[1]-004.001 1
0.457 294.1 0.200 233.9 0.75 117219[1]-005.001 1 0.768 418.8 1.149
786.9 0.05 117219[1]-007.001 1 0.536 380.0 0.473 256.0 0.05
117219[1]-009.001 2 9.9 105.4 2.3 116.2 0.1 117219[1]-012.001 2 7.5
67.6 3.0 177.3 0.05 117219[1]-013.001 2 18.6 257.8 4.5 97.7 0.1
117219[1]-015.001 2 11.7 173.6 1.8 108.4 0.1 117219[1]-017.001 2
7.2 91.1 3.6 73.0 0.01 117219[1]-018.001 2 15.2 167.7 3.1 181.0
0.25 117219[1]-020.001 2 8.5 136.2 1.9 116.2 0.25 117219[1]-022.001
2 13.1 237.2 24.7 639.1 0.75 117219[1]-023.001 2 10.7 149.1 18.0
522.8 0.05 117219[1]-031.001 2 13.9 250.7 10.9 134.4 0.1
117219[1]-033.001 2 8.3 112.2 2.1 166.6 0.5 117219[1]-037.001 2 6.0
82.1 1.7 96.3 0.1 117219[1]-038.001 2 9.8 147.0 4.8 151.2 0.5
117219[1]-040.001 2 10.1 118.6 2.4 29.7 0.1 *RTL = Relative
Transcript Level as measured against TIP4-like gene transcript
levels.
TABLE-US-00018 TABLE 13 Greenhouse bioassay and molecular analyses
results of negative control plants comprising transgenic and
nontransformed maize plants. Leaf Tissue Root Tissue PER5 PER5
Batch ST-LS1 UTR ST-LS1 UTR Root Sample ID # RTL* RTL RTL* RTL
Rating YFP protein Events 101556[691]-10720.001 1 0.000 75.1 0.000
56.1 1 101556[691]-10721.001 1 0.000 71.5 0.166 114.6 1
101556[691]-10722.001 1 0.000 259.6 0.000 0.0 **NG
101556[691]-10723.001 1 0.000 136.2 0.000 148.1 1
101556[691]-10724.001 1 0.000 82.1 0.000 16.9 1
101556[691]-10725.001 2 0.8 15.2 0.0 24.9 1 101556[691]-10726.001 2
0.7 16.2 0.0 55.7 0.5 101556[691]-10727.001 2 1.2 32.0 0.0 24.8 0.5
101556[691]-10728.001 2 0.0 7.9 0.0 54.9 1 101556[691]-10729.001 2
0.0 16.9 0.0 23.6 1 101556[691]-10948.001 3 0.0 21.6 ***ND ***ND
0.75 101556[691]-10949.001 3 0.0 40.5 ***ND ***ND 0.75
101556[691]-10950.001 3 0.0 42.2 ***ND ***ND 1
101556[691]-10951.001 3 0.4 0.0 ***ND ***ND 1 101556[691]-10952.001
3 0.0 58.1 ***ND ***ND 1 YFP hairpin Events 110853[9]-336.001 1
0.000 0.5 0.000 0.6 0.75 110853[9]-337.001 1 1.064 526.4 0.000 1.5
1 110853[9]-338.001 1 0.536 219.8 0.707 108.4 1 110853[9]-339.001 1
0.000 0.0 0.000 0.6 1 110853[9]-340.001 2 2.7 25.1 7.5 61.8 1
110853[9]-341.001 2 3.5 45.6 2.2 24.1 1 110853[9]-343.001 2 3.6
62.2 6.6 68.6 1 110853[9]-344.001 2 3.5 58.9 4.7 31.8 0.5
110853[9]-345.001 2 3.1 42.5 5.6 40.5 1 110853[9]-346.001 3 0.0 0.0
***ND ***ND 1 110853[9]-347.001 3 0.0 0.1 ***ND ***ND 1
110853[9]-348.001 3 9.5 183.5 ***ND ***ND 0.5 Nontransformed Plants
7sh382 1 0.000 0.4 0.000 8.7 1 7sh382 1 0.000 0.3 0.000 2.3 1
7sh382 1 0.000 0.2 0.000 0.0 1 7sh382 1 0.000 0.2 0.000 4.4 0.75
7sh382 1 0.000 0.4 0.000 6.8 0.5 7sh382 2 0.0 0.1 0.0 34.8 1 7sh382
2 0.0 0.1 1.5 0.2 1 7sh382 2 0.4 0.1 ***ND ***ND 1 7sh382 2 ***ND
***ND 0.0 41.9 0.5 7sh382 2 1.1 0.2 0.0 2.1 1 7sh382 3 0.0 0.1
***ND ***ND 1 7sh382 3 0.0 0.1 ***ND ***ND 0.5 7sh382 3 0.6 0.1
***ND ***ND 1 7sh382 3 0.0 0.1 ***ND ***ND 1 7sh382 4 1.7 1.3 ***ND
***ND 0.75 7sh382 4 0.6 0.1 ***ND ***ND 1 7sh382 4 0.0 0.1 ***ND
***ND 1 7sh382 4 0.7 0.1 ***ND ***ND 1 7sh382 4 0.0 0.0 ***ND ***ND
1 B104 1 0.000 0.0 0.000 1.9 1 B104 1 0.000 0.1 0.000 99.0 1 B104 1
0.000 1.1 0.000 7.1 1 B104 1 0.000 0.1 0.000 31.6 1 B104 1 0.000
0.0 0.000 2.3 1 B104 2 0.0 0.1 0.9 0.1 1 B104 2 0.3 3.6 0.0 4.3 1
B104 2 2.4 16.8 0.3 0.5 1 B104 2 0.0 0.1 0.8 0.0 1 B104 3 0.0 0.0
***ND ***ND 1 B104 3 0.0 0.0 ***ND ***ND 1 B104 3 0.0 0.0 ***ND
***ND 1 B104 3 0.0 0.1 ***ND ***ND 1 B104 4 0.3 0.0 ***ND ***ND 1
B104 4 0.4 0.0 ***ND ***ND 1 B104 4 0.0 0.0 ***ND ***ND 1 B104 4
0.5 0.0 ***ND ***ND 1 B104 4 0.0 0.2 ***ND ***ND 1 *RTL = Relative
Transcript Level as measured against TIP4-like gene transcript
levels. **NG = Not Graded due to small plant size. ***ND = Not
Done.
Example 9
Transgenic Zea mays Comprising Coleopteran Pest Sequences
[0334] Ten to 20 transgenic T.sub.0 Zea mays plants are generated
as described in EXAMPLE 6. A further 10-20 T.sub.1 Zea mays
independent lines expressing hairpin dsRNA for an RNAi construct
are obtained for corn rootworm challenge. Hairpin dsRNA may be
derived as set forth in SEQ ID NO:8 or otherwise further comprising
SEQ ID NO:1. Additional hairpin dsRNAs may be derived, for example,
from coleopteran pest sequences such as, for example, Caf1-180
(U.S. Patent Application Publication No. 2012/0174258), VatpaseC
(U.S. Patent Application Publication No. 2012/0174259), Rho1 (U.S.
Patent Application Publication No. 2012/0174260), VatpaseH (U.S.
Patent Application Publication No. 2012/0198586), PPI-87B (U.S.
Patent Application Publication No. 2013/0091600), RPA70 (U.S.
Patent Application Publication No. 2013/0091601), or RPS6 (U.S.
Patent Application Publication No. 2013/0097730). These are
confirmed through RT-PCR or other molecular analysis methods. Total
RNA preparations from selected independent T.sub.1 lines are
optionally used for RT-PCR with primers designed to bind in the
ST-LS1 intron of the hairpin expression cassette in each of the
RNAi constructs. In addition, specific primers for each target gene
in an RNAi construct are optionally used to amplify and confirm the
production of the pre-processed mRNA required for siRNA production
in planta. The amplification of the desired bands for each target
gene confirms the expression of the hairpin RNA in each transgenic
Zea mays plant. Processing of the dsRNA hairpin of the target genes
into siRNA is subsequently optionally confirmed in independent
transgenic lines using RNA blot hybridizations.
[0335] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect corn rootworms in
a way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development and viability of feeding coleopteran pests.
[0336] In planta delivery of dsRNA, siRNA or miRNA corresponding to
target genes and the subsequent uptake by coleopteran pests through
feeding results in down-regulation of the target genes in the
coleopteran pest through RNA-mediated gene silencing. When the
function of a target gene is important at one or more stages of
development, the growth, development, and reproduction of the
coleopteran pest is affected, and in the case of at least one of
WCR, NCR, SCR, MCR, D. balteata LeConte, D. u. tenella, and D. u.
undecimpunctata Mannerheim, leads to failure to successfully
infest, feed, develop, and/or reproduce, or leads to death of the
coleopteran pest. The choice of target genes and the successful
application of RNAi is then used to control coleopteran pests.
[0337] Phenotypic Comparison of Transgenic RNAi Lines and
Nontransformed Zea mays.
[0338] Target coleopteran pest genes or sequences selected for
creating hairpin dsRNA have no similarity to any known plant gene
sequence. Hence it is not expected that the production or the
activation of (systemic) RNAi by constructs targeting these
coleopteran pest genes or sequences will have any deleterious
effect on transgenic plants. However, development and morphological
characteristics of transgenic lines are compared with
nontransformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and nontransformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 10
Transgenic Zea mays Comprising a Coleopteran Pest Sequence and
Additional RNAi Constructs
[0339] A transgenic Zea mays plant comprising a heterologous coding
sequence in its genome that is transcribed into an iRNA molecule
that targets an organism other than a coleopteran pest is
secondarily transformed via Agrobacterium or WHISKERS.TM.
methodologies (see Petolino and Arnold (2009) Methods Mol. Biol.
526:59-67) to produce one or more insecticidal dsRNA molecules (for
example, at least one dsRNA molecule including a dsRNA molecule
targeting a gene comprising SEQ ID NO:1). Plant transformation
plasmid vectors prepared essentially as described in EXAMPLE 4 are
delivered via Agrobacterium or WHISKERS.TM.-mediated transformation
methods into maize suspension cells or immature maize embryos
obtained from a transgenic Hi II or B104 Zea mays plant comprising
a heterologous coding sequence in its genome that is transcribed
into an iRNA molecule that targets an organism other than a
coleopteran pest.
Example 11
Transgenic Zea mays Comprising an RNAi Construct and Additional
Coleopteran Pest Control Sequences
[0340] A transgenic Zea mays plant comprising a heterologous coding
sequence in its genome that is transcribed into an iRNA molecule
that targets a coleopteran pest organism (for example, at least one
dsRNA molecule including a dsRNA molecule targeting a gene
comprising SEQ ID NO:1) is secondarily transformed via
Agrobacterium or WHISKERS.TM. methodologies (see Petolino and
Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one or more
insecticidal protein molecules, for example, Cry1B, Cry1I, Cry2A,
Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35,
Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C insecticidal proteins.
Plant transformation plasmid vectors prepared essentially as
described in EXAMPLE 4 are delivered via Agrobacterium or
WHISKERS.TM.-mediated transformation methods into maize suspension
cells or immature maize embryos obtained from a transgenic B104 Zea
mays plant comprising a heterologous coding sequence in its genome
that is transcribed into an iRNA molecule that targets a
coleopteran pest organism. Doubly-transformed plants are obtained
that produce iRNA molecules and insecticidal proteins for control
of coleopteran pests.
Example 12
Mortality of Neotropical Brown Stink Bug (Euschistus heros)
Following COPI Beta RNAi Injection
[0341] Neotropical Brown Stink Bug (BSB; Euschistus heros) were
reared in a 27.degree. C. incubator, at 65% relative humidity, with
16:8 hour light:dark cycle. One gram of eggs collected over 2-3
days were seeded in 5 L containers with filter paper discs at the
bottom; the containers were covered with #18 mesh for ventilation.
Each rearing container yielded approximately 300-400 adult BSB. At
all stages, the insects were fed fresh green beans three times per
week, a sachet of seed mixture that contained sunflower seeds,
soybeans, and peanuts (3:1:1 by weight ratio) was replaced once a
week. Water was supplemented in vials with cotton plugs as a wicks.
After the initial two weeks, insects were transferred onto new
container once a week.
[0342] BSB Artificial Diet.
[0343] BSB artificial diet prepared as follows (used within two
weeks of preparation). Lyophilized green beans were blended to a
fine powder in a MAGIC BULLET.RTM. blender while raw (organic)
peanuts were blended in a separate MAGIC BULLET.RTM. blender.
Blended dry ingredients were combined (weight percentages: green
beans, 35%; peanuts, 35%; sucrose, 5%; Vitamin complex (e.g.
Vanderzant Vitamin Mixture for insects, SIGMA-ALDRICH, Catalog No.
V1007), 0.9%); in a large MAGIC BULLET.RTM. blender, which was
capped and shaken well to mix the ingredients. The mixed dry
ingredients were then added to a mixing bowl. In a separate
container, water and benomyl anti-fungal agent (50 ppm; 25 .mu.L of
a 20,000 ppm solution/50 mL diet solution) were mixed well and then
added to the dry ingredient mixture. All ingredients were mixed by
hand until the solution was fully blended. The diet was shaped into
desired sizes, wrapped loosely in aluminum foil, heated for 4 hours
at 60.degree. C., then cooled and stored at 4.degree. C.
[0344] RNAi Target Selection.
[0345] Six stages of BSB development were selected for mRNA library
preparation. Total RNA was extracted from insects frozen at
-70.degree. C. and homogenized in 10 volumes of Lysis/Binding
buffer in Lysing MATRIX A 2 mL tubes (MP BIOMEDICALS, Santa Ana,
Calif.) on a FastPrep.RTM.-24 Instrument (MP BIOMEDICALS). Total
mRNA was extracted using a mirVana.TM. miRNA Isolation Kit (AMBION;
INVITROGEN) according to the manufacturer's protocol. RNA
sequencing using an Illumina.RTM. HiSeg.TM. system (San Diego,
Calif.) provided candidate target gene sequences for use in RNAi
insect control technology. HiSeg.TM. generated a total of about 378
million reads for the six samples. The reads were assembled
individually for each sample using TRINITY assembler software
(Grabherr et al. (2011) Nature Biotech. 29:644-652). The assembled
transcripts were combined to generate a pooled transcriptome. This
BSB pooled transcriptome contains 378,457 sequences.
[0346] BSB COPI Beta Ortholog Identification.
[0347] A tBLASTn search of the BSB pooled transcriptome was
performed using as query sequence a Drosophila COPI beta protein
(SCOP-PA (GENBANK Accession No. NP_523400). BSB COPI beta (SEQ ID
NO:83 and SEQ ID NO:84 were identified as a Euschistus heros
candidate target gene.
[0348] The sequences SEQ ID NO:83 and SEQ ID NO:84 are novel.
Euschistus heros COPI BETA protein 2 (SEQ ID NO:95) have some
homology to SEQ ID NO:283 and SEQ ID NO: 275 disclosed in U.S.
Patent Application No. WO2012055982 and US20130291188. The
Euschistus COPI beta sequence (SEQ ID NO:83) is somewhat related
(74% identity) to a fragment of a COPI beta gene from the
Mediterranean fruit fly, Ceratitis capitata (GENBANK Accession No.
XM_004521524.1). The Euschistus COPI beta sequence (SEQ ID NO:84)
is somewhat related (74% identity) to a fragment of a COPI beta
gene from Apis dorsata (GENBANK Accession No. XM_006615233.1). The
closest homolog of the Euschistus heros COPI beta amino acid
sequence 1 (SEQ ID NO:94) is a Pediculus humanus corporis protein
having GENBANK Accession No. XP_002424856.1 (84% similar; 72%
identical over the homology region). The closest homolog of the
Euschistus heros COPI beta amino acid sequence 2 (SEQ ID NO:95) is
a Tribolium casetanum protein having GENBANK Accession No.
XP_001816488.1 (86% similar; 78% identical over the homology
region).
[0349] Template Preparation and dsRNA Synthesis.
[0350] cDNA was prepared from total BSB RNA extracted from a single
young adult insect (about 90 mg) using TRIzol.RTM. Reagent (LIFE
TECHNOLOGIES). The insect was homogenized at room temperature in a
1.5 mL microcentrifuge tube with 200 .mu.L of TRIzol.RTM. using a
pellet pestle (FISHERBRAND Catalog No. 12-141-363) and Pestle Motor
Mixer (COLE-PARMER, Vernon Hills, Ill.). Following homogenization,
an additional 800 .mu.L of TRIzol.RTM. was added, the homogenate
was vortexed, and then incubated at room temperature for five
minutes. Cell debris was removed by centrifugation and the
supernatant was transferred to a new tube. 200 .mu.L of chloroform
were added and the mixture was vortexed for 15 seconds. After
allowing the extraction to sit at room temperature for 2 to 3 min,
the phases were separated by centrifugation at 12,000.times.g at
4.degree. C. for 15 minutes. The upper aqueous phase was carefully
transferred into another nuclease-free 1.5 mL microcentrifuge tube,
and the RNA was precipitated with 500 .mu.L of room temperature
isopropanol. After ten-minute incubation at room temperature, the
mixture was centrifuged for 10 minutes as above. The RNA pellet was
rinsed with 1 mL of room-temperature 75% ethanol and centrifuged
for an additional 10 minutes as above. Following
manufacturer-recommended TRIzol.RTM. extraction protocol for 1 mL
of TRIzol.RTM., the RNA pellet was dried at room temperature and
resuspended in 200 .mu.L of Tris Buffer from a GFX PCR DNA AND GEL
EXTRACTION KIT (Illustra.TM.; GE HEALTHCARE LIFE SCIENCES) using
Elution Buffer Type 4 (i.e. 10 mM Tris-HCl pH8.0). RNA
concentration was determined using a NANODROP.TM. 8000
spectrophotometer (THERMO SCIENTIFIC, Wilmington, Del.).
[0351] cDNA was reverse-transcribed from 5 .mu.g of BSB total RNA
template and oligo dT primer using a SUPERSCRIPT III FIRST-STRAND
SYNTHESIS SYSTEM.TM. for RT-PCR (INVITROGEN), following the
supplier's recommended protocol. The final volume of the
transcription reaction was brought to 100 .mu.L with nuclease-free
water.
[0352] Primers BSB_.beta.COP-1-For (SEQ ID NO:87) and
BSB_.beta.COP-1-Rev (SEQ ID NO:88) were used to amplify BSB_COPI
beta-1 template. Primers BSB_.beta.COP-2-For (SEQ ID NO:89) and
BSB_.beta.COP-2-Rev (SEQ ID NO:90) were used to amplify BSB_COPI
beta-2 template. The DNA templates were amplified by touch-down PCR
(annealing temperature lowered from 60.degree. C. to 50.degree. C.
in a 1.degree. C./cycle decrease) with 1 .mu.L of cDNA (above) as
the template. Fragments comprising 498 bp segment of COPI beta:
BSB_COPI beta region1, also referred to as BSB_COPI beta-1 (SEQ ID
NO:85), and 441 bp segments of COPI beta: BSB_COPI beta region 2,
also referred to as BSB_COPI beta-2 (SEQ ID NO:86) were generated
during 35 cycles of PCR. The above procedure was also used to
amplify a 301 bp negative control template YFPv2 (SEQ ID NO:91)
using YFPv2-F (SEQ ID NO:92) and YFPv2-R (SEQ ID NO:93) primers.
The BSB_COPI beta and YFPv2 primers contained a T7 phage promoter
sequence (SEQ ID NO:4) at their 5' ends, and thus enabled the use
of YFPv2, BSB_COPI beta-1, and BSB_COPI beta-2 DNA fragments for
dsRNA transcription.
[0353] dsRNA was synthesized using 2 .mu.L of PCR product (above)
as the template with a MEGAscript.TM. RNAi kit (AMBION) used
according to the manufacturer's instructions. (See FIG. 1). dsRNA
was quantified on a NANODROP.TM. 8000 spectrophotometer and diluted
to 500 ng/.mu.L in nuclease-free 0.1.times.TE buffer (1 mM Tris
HCL, 0.1 mM EDTA, pH7.4).
[0354] Injection of dsRNA into BSB hemoceol BSB were reared on
artificial diet (above) in a 27.degree. C. incubator at 65%
relative humidity and 16:8 hour light:dark photoperiod. Second
instar nymphs (each weighing 1 to 1.5 mg) were gently handled with
a small brush to prevent injury and were placed in a Petri dish on
ice to chill and immobilize the insects. Each insect was injected
with 55.2 nL of a 500 ng/.mu.L dsRNA solution (i.e. 27.6 ng dsRNA;
dosage of 18.4 to 27.6 .mu.g/g body weight). Injections were
performed using a NANOJECT.TM. II injector (DRUMMOND SCIENTIFIC,
Broomhall, Pa.) equipped with an injection needle pulled from a
Drummond 3.5 inch #3-000=203-G/X glass capillary. The needle tip
was broken and the capillary was backfilled with light mineral oil,
then filled with 2 to 3 .mu.L of dsRNA. dsRNA was injected into the
abdomen of the nymphs (10 insects injected per dsRNA per trial),
and the trials were repeated on three different days. Injected
insects (5 per well) were transferred into 32-well trays (Bio-RT-32
Rearing Tray; BIO-SERV, Frenchtown, N.J.) containing a pellet of
artificial BSB diet and covered with Pull-N-Peel.TM. tabs
(BIO-CV-4; BIO-SERV). Moisture was supplied by means of 1.25 mL of
water in a 1.5 mL microcentrifuge tube with a cotton wick. The
trays were incubated at 26.5.degree. C., 60% humidity and 16:8
light:dark photoperiod. Viability counts and weights were taken on
day 7 after the injections.
[0355] Injections Identified BSB COPI Beta as a Lethal dsRNA
[0356] dsRNA that targets segment of YFP coding region, YFPv2 was
used as a negative control in BSB injection experiments. As
summarized in Table 14, 27.6 ng of BSB_COPI beta-1, and BSB_COPI
beta-2 dsRNA injected into the hemoceol of 2.sup.nd instar BSB
nymphs produced high mortality within seven days. The mortality
caused by both BSB_COPI beta-1 or BSB_COPI beta-2 dsRNA was
numerically higher than the base-level mortality observed with
YFPv2 dsRNA (negative control), although only BSB_COPI beta-2 was
statistically significant (Student's t-test).
TABLE-US-00019 TABLE 14 Results of BSB_COPI beta-1 or BSB_COPI
beta-2 dsRNA injection into the hemoceol of 2.sup.nd instar Brown
Stink Bug nymphs seven days after injection. Mean % Mortality .+-.
p value Treatment* N Trials SEM t-test BSB COPI beta-1 3 66.7 .+-.
20.3 1.12E-01 BSB COPI beta-2 3 76.7 .+-. 14.5 2.89E-02 YFP v2
dsRNA 3 23.3 .+-. 6.67 *Ten insects injected per trial for each
dsRNA.
Example 13
Transgenic Zea mays Comprising Hemipteran Pest Sequences
[0357] Ten to 20 transgenic T.sub.0 Zea mays plants harboring
expression vectors for nucleic acids comprising SEQ ID NO: 83, SEQ
ID NO:84, SEQ ID NO:85 and/or SEQ ID NO:86 are generated as
described in EXAMPLE 6. A further 10-20 T.sub.1 Zea mays
independent lines expressing hairpin dsRNA for an RNAi construct
are obtained for BSB challenge. Hairpin dsRNA may be derived as set
forth in SEQ ID NO:85 and/or SEQ ID NO:86 or otherwise further
comprising SEQ ID NO:83 and/or SEQ ID NO:84. These are confirmed
through RT-PCR or other molecular analysis methods. Total RNA
preparations from selected independent T.sub.1 lines are optionally
used for RT-PCR with primers designed to bind in the ST-LS1 intron
of the hairpin expression cassette in each of the RNAi constructs.
In addition, specific primers for each target gene in an RNAi
construct are optionally used to amplify and confirm the production
of the pre-processed mRNA required for siRNA production in planta.
The amplification of the desired bands for each target gene
confirms the expression of the hairpin RNA in each transgenic Zea
mays plant. Processing of the dsRNA hairpin of the target genes
into siRNA is subsequently optionally confirmed in independent
transgenic lines using RNA blot hybridizations.
[0358] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect corn rootworms in
a way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development and viability of feeding hemipteran pests.
[0359] In planta delivery of dsRNA, siRNA, shRNA, or miRNA
corresponding to target genes and the subsequent uptake by
hemipteran pests through feeding results in down-regulation of the
target genes in the hemipteran pest through RNA-mediated gene
silencing. When the function of a target gene is important at one
or more stages of development, the growth, development, and
reproduction of the hemipteran pest is affected, and in the case of
at least one of Euschistus heros, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Acrosternum hilare, and Euschistus servus
leads to failure to successfully infest, feed, develop, and/or
reproduce, or leads to death of the hemipteran pest. The choice of
target genes and the successful application of RNAi is then used to
control hemipteran pests.
[0360] Phenotypic Comparison of Transgenic RNAi Lines and
Nontransformed Zea mays
[0361] Target hemipteran pest genes or sequences selected for
creating hairpin dsRNA have no similarity to any known plant gene
sequence. Hence it is not expected that the production or the
activation of (systemic) RNAi by constructs targeting these
hemipteran pest genes or sequences will have any deleterious effect
on transgenic plants. However, development and morphological
characteristics of transgenic lines are compared with
nontransformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and nontransformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 14
Transgenic Glycine max Comprising Hemipteran Pest Sequences
[0362] Ten to 20 transgenic T.sub.0 Glycine max plants harboring
expression vectors for nucleic acids comprising SEQ ID NO: 83, SEQ
ID NO:84, SEQ ID NO:85 and/or SEQ ID NO:86 are generated as is
known in the art, including for example by Agrobacterium-mediated
transformation, as follows. Mature soybean (Glycine max) seeds are
sterilized overnight with chlorine gas for sixteen hours. Following
sterilization with chlorine gas, the seeds are placed in an open
container in a LAMINAR.TM. flow hood to dispel the chlorine gas.
Next, the sterilized seeds are imbibed with sterile H.sub.2O for
sixteen hours in the dark using a black box at 24.degree. C.
[0363] Preparation of Split-Seed Soybeans.
[0364] The split soybean seed comprising a portion of an embryonic
axis protocol required preparation of soybean seed material which
is cut longitudinally, using a #10 blade affixed to a scalpel,
along the hilum of the seed to separate and remove the seed coat,
and to split the seed into two cotyledon sections. Careful
attention is made to partially remove the embryonic axis, wherein
about 1/2-1/3 of the embryo axis remains attached to the nodal end
of the cotyledon.
[0365] Inoculation.
[0366] The split soybean seeds comprising a partial portion of the
embryonic axis are then immersed for about 30 minutes in a solution
of Agrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105)
containing binary plasmid comprising SEQ ID NO: 83, SEQ ID NO:84,
SEQ ID NO:85 and/or SEQ ID NO:86. The Agrobacterium tumefaciens
solution is diluted to a final concentration of .lamda.=0.6 OD650
before immersing the cotyledons comprising the embryo axis.
[0367] Co-Cultivation.
[0368] Following inoculation, the split soybean seed is allowed to
co-cultivate with the Agrobacterium tumefaciens strain for 5 days
on co-cultivation medium (Wang, Kan. Agrobacterium Protocols. 2. 1.
New Jersey: Humana Press, 2006. Print.) in a Petri dish covered
with a piece of filter paper.
[0369] Shoot Induction.
[0370] After 5 days of co-cultivation, the split soybean seeds are
washed in liquid Shoot Induction (SI) media consisting of B5 salts,
B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na2EDTA, 30 g/L sucrose, 0.6
g/L MES, 1.11 mg/L BAP, 100 mg/L Timentin.TM., 200 mg/L cefotaxime,
and 50 mg/L vancomycin (pH 5.7). The split soybean seeds are then
cultured on Shoot Induction I (SI I) medium consisting of B5 salts,
B5 vitamins, 7 g/L Noble agar, 28 mg/L Ferrous, 38 mg/L Na2EDTA, 30
g/L sucrose, 0.6 g/L MES, 1.11 mg/L BAP, 50 mg/L Timentin.TM., 200
mg/L cefotaxime, 50 mg/L vancomycin (pH 5.7), with the flat side of
the cotyledon facing up and the nodal end of the cotyledon imbedded
into the medium. After 2 weeks of culture, the explants from the
transformed split soybean seed are transferred to the Shoot
Induction II (SI II) medium containing SII medium supplemented with
6 mg/L glufosinate (Liberty.RTM.).
[0371] Shoot Elongation.
[0372] After 2 weeks of culture on SI II medium, the cotyledons are
removed from the explants and a flush shoot pad containing the
embryonic axis are excised by making a cut at the base of the
cotyledon. The isolated shoot pad from the cotyledon is transferred
to Shoot Elongation (SE) medium. The SE medium consists of MS
salts, 28 mg/L Ferrous, 38 mg/L Na2EDTA, 30 g/L sucrose and 0.6 g/L
MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid, 0.1 mg/L
IAA, 0.5 mg/L GA3, 1 mg/L zeatin riboside, 50 mg/L Timentin.TM.,
200 mg/L cefotaxime, 50 mg/L vancomycin, 6 mg/L glufosinate, 7 g/L
Noble agar, (pH 5.7). The cultures are transferred to fresh SE
medium every 2 weeks. The cultures are grown in a Conviron.TM.
growth chamber at 24.degree. C. with an 18 h photoperiod at a light
intensity of 80-90 mol/m.sup.2 sec.
[0373] Rooting.
[0374] Elongated shoots which developed from the cotyledon shoot
pad are isolated by cutting the elongated shoot at the base of the
cotyledon shoot pad, and dipping the elongated shoot in 1 mg/L IBA
(Indole 3-butyric acid) for 1-3 minutes to promote rooting. Next,
the elongated shoots are transferred to rooting medium (MS salts,
B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na2EDTA, 20 g/L sucrose and
0.59 g/L MES, 50 mg/L asparagine, 100 mg/L L-pyroglutamic acid 7
g/L Noble agar, pH 5.6) in phyta trays.
[0375] Cultivation.
[0376] Following culture in a Conviron.TM. growth chamber at
24.degree. C., 18 h photoperiod, for 1-2 weeks, the shoots which
have developed roots are transferred to a soil mix in a covered
sundae cup and placed in a Conviron.TM. growth chamber (models
CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg,
Manitoba, Canada) under long day conditions (16 hours light/8 hours
dark) at a light intensity of 120-150 .mu.mol/m.sup.2 sec under
constant temperature (22.degree. C.) and humidity (40-50%) for
acclimatization of plantlets. The rooted plantlets are acclimated
in sundae cups for several weeks before they are transferred to the
greenhouse for further acclimatization and establishment of robust
transgenic soybean plants.
[0377] A further 10-20 T.sub.1 Glycine max independent lines
expressing hairpin dsRNA for an RNAi construct are obtained for BSB
challenge. Hairpin dsRNA may be derived as set forth in SEQ ID
NO:85, SEQ ID NO:86 or otherwise further comprising SEQ ID NO:83
and/or SEQ ID NO:84. These are confirmed through RT-PCR or other
molecular analysis methods. Total RNA preparations from selected
independent T.sub.1 lines are optionally used for RT-PCR with
primers designed to bind in the ST-LS1 intron of the hairpin
expression cassette in each of the RNAi constructs. In addition,
specific primers for each target gene in an RNAi construct are
optionally used to amplify and confirm the production of the
pre-processed mRNA required for siRNA production in planta. The
amplification of the desired bands for each target gene confirms
the expression of the hairpin RNA in each transgenic Glycine max
plant. Processing of the dsRNA hairpin of the target genes into
siRNA is subsequently optionally confirmed in independent
transgenic lines using RNA blot hybridizations.
[0378] Moreover, RNAi molecules having mismatch sequences with more
than 80% sequence identity to target genes affect corn rootworms in
a way similar to that seen with RNAi molecules having 100% sequence
identity to the target genes. The pairing of mismatch sequence with
native sequences to form a hairpin dsRNA in the same RNAi construct
delivers plant-processed siRNAs capable of affecting the growth,
development and viability of feeding hemipteran pests.
[0379] In planta delivery of dsRNA, siRNA, shRNA, or miRNA
corresponding to target genes and the subsequent uptake by
hemipteran pests through feeding results in down-regulation of the
target genes in the hemipteran pest through RNA-mediated gene
silencing. When the function of a target gene is important at one
or more stages of development, the growth, development, and
reproduction of the hemipteran pest is affected, and in the case of
at least one of Euschistus heros, Piezodorus guildinii, Halyomorpha
halys, Nezara viridula, Acrosternum hilare, and Euschistus servus
leads to failure to successfully infest, feed, develop, and/or
reproduce, or leads to death of the hemipteran pest. The choice of
target genes and the successful application of RNAi is then used to
control hemipteran pests.
[0380] Phenotypic comparison of transgenic RNAi lines and
nontransformed Glycine max Target hemipteran pest genes or
sequences selected for creating hairpin dsRNA have no similarity to
any known plant gene sequence. Hence it is not expected that the
production or the activation of (systemic) RNAi by constructs
targeting these hemipteran pest genes or sequences will have any
deleterious effect on transgenic plants. However, development and
morphological characteristics of transgenic lines are compared with
nontransformed plants, as well as those of transgenic lines
transformed with an "empty" vector having no hairpin-expressing
gene. Plant root, shoot, foliage and reproduction characteristics
are compared. There is no observable difference in root length and
growth patterns of transgenic and nontransformed plants. Plant
shoot characteristics such as height, leaf numbers and sizes, time
of flowering, floral size and appearance are similar. In general,
there are no observable morphological differences between
transgenic lines and those without expression of target iRNA
molecules when cultured in vitro and in soil in the glasshouse.
Example 15
E. heros Bioassays on Artificial Diet
[0381] In dsRNA feeding assays on artificial diet, 32-well trays
are set up with an .about.18 mg pellet of artificial diet and
water, as for injection experiments (EXAMPLE 12). dsRNA at a
concentration of 200 ng/.mu.l is added to the food pellet and water
sample, 100 .mu.l to each of two wells. Five 2.sup.nd instar E.
heros nymphs are introduced into each well. Water samples and dsRNA
that targets YFP transcript are used as negative controls. The
experiments are repeated on three different days. Surviving insects
are weighed and the mortality rates are determined after 8 days of
treatment.
Example 16
Transgenic Arabidopsis thaliana Comprising Hemipteran Pest
Sequences
[0382] Arabidopsis transformation vectors containing a target gene
construct for hairpin formation comprising segments of COPI beta
(SEQ ID NO:83 and/or SEQ ID NO:84) are generated using standard
molecular methods similar to EXAMPLE 4. Arabidopsis transformation
is performed using standard Agrobacterium-based procedure. T.sub.1
seeds are selected with glufosinate tolerance selectable marker.
Transgenic T.sub.1 Arabidopsis plants are generated and homozygous
simple-copy T.sub.2 transgenic plants are generated for insect
studies. Bioassays are performed on growing Arabidopsis plants with
inflorescences. Five to ten insects are placed on each plant and
monitored for survival within 14 days.
[0383] Construction of Arabidopsis Transformation Vectors.
[0384] Entry clones based on entry vector pDAB3916 harboring a
target gene construct for hairpin formation comprising a segment of
COPI beta (SEQ ID NO:83 and/or SEQ ID NO:84) are assembled using a
combination of chemically synthesized fragments (DNA2.0, Menlo
Park, Calif.) and standard molecular cloning methods.
Intramolecular hairpin formation by RNA primary transcripts is
facilitated by arranging (within a single transcription unit) two
copies of a target gene segment in opposite orientations, the two
segments being separated by an ST-LS1 intron sequence (SEQ ID
NO:10) (Vancanneyt et al. (1990) Mol. Gen. Genet. 220(2):245-50).
Thus, the primary mRNA transcript contains the two COPI beta gene
segment sequences as large inverted repeats of one another,
separated by the intron sequence. A copy of a Arabidopsis thaliana
ubiquitin 10 promoter (Callis et al. (1990) J. Biological Chem.
265:12486-12493) is used to drive production of the primary mRNA
hairpin transcript, and a fragment comprising a 3' untranslated
region from Open Reading Frame 23 of Agrobacterium tumefaciens
(AtuORF23 3' UTR v1; U.S. Pat. No. 5,428,147) is used to terminate
transcription of the hairpin-RNA-expressing gene.
[0385] The hairpin clone within entry vector pDAB3916 described
above is used in standard GATEWAY.RTM. recombination reaction with
a typical binary destination vector pDAB101836 to produce hairpin
RNA expression transformation vectors for Agrobacterium-mediated
Arabidopsis transformation.
[0386] Binary destination vector pDAB101836 comprises a herbicide
tolerance gene, DSM-2v2 (U.S. Patent App. No. 2011/0107455), under
the regulation of a Cassava vein mosaic virus promoter (CsVMV
Promoter v2, U.S. Pat. No. 7,601,885; Verdaguer et al, (1996) Plant
Molecular Biology, 31:1129-1139). A fragment comprising a 3'
untranslated region from Open Reading Frame 1 of Agrobacterium
tumefaciens (AtuORF1 3' UTR v6; Huang et al, (1990) J. Bacteriol,
172:1814-1822) is used to terminate transcription of the DSM2v2
mRNA.
[0387] A negative control binary construct, pDAB114507, which
comprises a gene that expresses a YFP hairpin RNA, is constructed
by means of standard GATEWAY.RTM. recombination reactions with a
typical binary destination vector (pDAB101836) and entry vector
pDAB3916. Entry construct pDAB112644 comprises a YFP hairpin
sequence (hpYFP v2-1, SEQ ID NO:96) under the expression control of
an Arabidopsis Ubiquitin 10 promoter (as above) and a fragment
comprising an ORF23 3' untranslated region from Agrobacterium
tumefaciens (as above).
[0388] Production of Transgenic Arabidopsis Comprising Insecticidal
Hairpin RNAs: Agrobacterium-Mediated Transformation.
[0389] Binary plasmids containing hairpin sequences are
electroporated into Agrobacterium strain GV3101 (pMP90RK). The
recombinant Agrobacterium clones are confirmed by restriction
analysis of plasmids preparations of the recombinant Agrobacterium
colonies. A Qiagen Plasmid Max Kit (Qiagen, Cat#12162) is used to
extract plasmids from Agrobacterium cultures following the
manufacture recommended protocol.
[0390] Arabidopsis Transformation and T.sub.1 Selection.
[0391] Twelve to fifteen Arabidopsis plants (c.v. Columbia) are
grown in 4'' pots in the green house with light intensity of 250
.mu.mol/m.sup.2, 25.degree. C., 18:6 hours of light:dark
conditions. Primary flower stems are trimmed one week before
transformation. Agrobacterium inoculums are prepared by incubating
10 .mu.l of recombinant Agrobacterium glycerol stock in 100 ml LB
broth (Sigma L3022)+100 mg/L Spectinomycin+50 mg/L Kanamycin at
28.degree. C. and shaking at 225 rpm for 72 hours. Agrobacterium
cells are harvested and suspended into 5% sucrose+0.04% Silwet-L77
(Lehle Seeds Cat # VIS-02)+10 .mu.g/L benzamino purine (BA)
solution to OD600 0.8.about.1.0 before floral dipping. The
above-ground parts of the plant are dipped into the Agrobacterium
solution for 5-10 minutes, with gentle agitation. The plants are
then transferred to the greenhouse for normal growth with regular
watering and fertilizing until seed set.
Example 17
Growth and Bioassays of Transgenic Arabidopsis
[0392] Selection of T.sub.1 Arabidopsis Transformed with Hairpin
RNAi Constructs.
[0393] Up to 200 mg of T.sub.1 seeds from each transformation is
stratified in 0.1% agarose solution. The seeds are planted in
germination trays (10.5''.times.21''.times.1''; T.O. Plastics Inc.,
Clearwater, Minn.) with #5 sunshine media. Transformants are
selected for tolerance to Ignite.RTM. (glufosinate) at 280 g/ha at
6 and 9 days post planting. Selected events are transplanted into
4'' diameter pots. Insertion copy analysis is performed within a
week of transplanting via hydrolysis quantitative Real-Time PCR
(qPCR) using Roche LightCycler480. The PCR primers and hydrolysis
probes are designed against DSM2v2 selectable marker using
LightCycler Probe Design Software 2.0 (Roche). Plants are
maintained at 24.degree. C., with a 16:8 hour light:dark
photoperiod under fluorescent and incandescent lights at intensity
of 100-150 mE/m2.times.s.
[0394] E. heros Plant Feeding Bioassay.
[0395] At least four low copy (1-2 insertions), four medium copy
(2-3 insertions), and four high copy (.gtoreq.4 insertions) events
are selected for each construct. Plants are grown to a flowering
stage (plants containing flowers and siliques). The surface of soil
is covered with .about.50 ml volume of white sand for easy insect
identification. Five to ten 2nd instar E. heros nymphs are
introduced onto each plant. The plants are covered with plastic
tubes that are 3'' in diameter, 16'' tall, and with wall thickness
of 0.03'' (Item No. 484485, Visipack Fenton Mo.); the tubes are
covered with nylon mesh to isolate the insects. The plants are kept
under normal temperature, light, and watering conditions in a
conviron. In 14 days, the insects are collected and weighed;
percent mortality as well as growth inhibition (1-weight
treatment/weight control) are calculated. YFP hairpin-expressing
plants are used as controls.
[0396] T2 Arabidopsis Seed Generation and T2 Bioassays.
[0397] T2 seed is produced from selected low copy (1-2 insertions)
events for each construct. Plants (homozygous and/or heterozygous)
are subjected to E. heros feeding bioassay, as described above. T3
seed is harvested from homozygotes and stored for future
analysis.
[0398] While the present disclosure may be susceptible to various
modifications and alternative forms, specific embodiments have been
described by way of example in detail herein. However, it should be
understood that the present disclosure is not intended to be
limited to the particular forms disclosed. Rather, the present
disclosure is to cover all modifications, equivalents, and
alternatives falling within the scope of the present disclosure as
defined by the following appended claims and their legal
equivalents.
Sequence CWU 1
1
9613392DNADiabrotica virgifera 1ccgatgaccg cccatgtcac tgtatttctc
aataggagaa gactgaacaa agagaagaaa 60ttagtcaagt catttgtcaa cgtcaactgt
aacatacgtc gcatcaaaac gtcaacaatc 120tgacacgtct gtaatgttta
gcctgacttt tgagtaaaat taccgaaaaa cataattaaa 180attgatttat
taagagtaca taataacgta cgaaaatgac cgcggtagaa caaccttgtt
240acacactaat aaacttgcca acagattcgg agccctacaa tgaaatgcaa
ctaaaaatgg 300atttagaaaa gggtgaggtt aaagtaaaaa taagagcatt
agaaaaaata attcacatga 360ttctggcagg agaaaggttg ccgaatggat
tcctaatgac cattataaga aacgttttac 420ctctacaaga tcatttggca
aaaaaactat tattgatttt ctgggaaata gttccaaaaa 480caaatccaga
aggtaaacta ctacaagaga tgattttggt atgtgatgcc tatagaaaag
540atctgcaaca cccaaatgaa tttttgagag gttctacact tcgcttcttg
tgcaaactga 600aggaaccaga attgttggaa ccattaatgc ccagtattag
agcttgtttg gatcataggc 660acagctatgt gaggaggaat gctgtactgg
caatttttac catttacaaa aattttgaag 720ccctcattcc agatgctcct
gaactgatct ccaattattt ggatggtgag caagacatgt 780cttgtaaaag
aaatgcgttt ttaatgcttc ttcatgctga ccaagaaagg gcgttgtcgt
840atttggcatc atgtttagat caagtaaatt catttggaga tattctacaa
ctggtcatcg 900ttgagttgat atataaggtg tgtcattcca atcctgcgga
aagatctaga tttattagat 960gtatatataa cttgttgaac tcaagcagtc
ctgctgtcag gtacgaagct gcaggaactt 1020tagtcaccct ctccagtgcc
ccgactgccg ttaaagctgc tgctagctgt tacattgagt 1080taattatcaa
agaaagtgac aacaatgtaa aactcatcgt tttggacagg ctgatagcac
1140ttaaggagct tcctaatcac gaaagaattc tgcaggattt agttatggac
atactgagag 1200tactctctgc tcctgactta gaagtccgca agaagacttt
aagtctagcc cttgaattag 1260tctcttcacg gaacatagaa gaaatggtat
tagtattaac aaaggaagtg agtaaaacgg 1320tagacagtga acatgaggat
acaggaaagt acaggcaatt gttagtaagg actctacatt 1380cgtgttccat
taagttccca gatatcgcac gtagtgttat accagtcttg attgaatttt
1440tatccgataa taatgaactg gctgccacag atgtattgct gttcttaagg
gaagccatac 1500agaagtttaa agaattgcaa ccgttaatta ttgagaaact
catcgaaact ttcaaagaca 1560ttaaattggt caaagtccat agagcagcaa
tttggatttt gggagaatac gcgagtactg 1620cttccgatat agaagttatc
gttggagaaa ttaacagatt gttgggtgaa ggatccctcg 1680ttgaagctga
gcagaagtta atagcaggag atacggaaga gaatgctcct gcacctgctg
1740caggcgccac cactttagtt acttccgatg gaacatatgc tacccaatca
gctttcaaca 1800ctgtcagcca aaccactaaa gaagcacgac ctcctctaag
acaatacctc atggatggtg 1860attttttcat cggagcctct ttggcatcta
cattaaccaa actgtctttg cggtatgagg 1920acctcacctc tcctgctgct
agcaatggat tcaatgccaa aattatgctt attatggctg 1980gaattcttca
cttgggaaaa tcaggacttc ccacaaaatc aataaccaac gacgataaag
2040accacattct gttctgttta cgagtcctat ctgatcgttc tccaatcatt
gttgaaattt 2100tcaaaaaatt gtgccgctcg gcactaaatg agatgcttct
agctaaggaa tcggtagaag 2160cgatctcgca aaagagcaaa gaaaaaaaca
agcgtacgat tcaaactgac gacgctataa 2220gcttcctgca attagagaca
gataaaagtg gagagctagg agaaaacgta ttcgagatgt 2280cgctgtcaca
agctttagta ggaggtcgaa cgggaggtgg cgaatcagta ttaagttcca
2340ataaattaga taaaatcaca caactgactg gtttttccga tccagtttat
tccgaagcat 2400acgttcacgt gaatcagtac gatatcgtgc ttgatgtctt
aatcgtaaac caaactaacg 2460atactttaca aaactgcacg ctagagctgg
ctactttagg cgatttgaag ttggtagaga 2520agccacaacc tgtcgtattg
gcgcccaaag acttttgcaa cattaaagct aacgtgaaag 2580tggcctcaac
tgaaaacgga attatatttg gcaacattgt gtatgatgtc ataggagcgg
2640ggtcagatag gaatgttgta gttttgaatg atatacacat agatataatg
gactatatag 2700tgcctgctag ttgtacagat agcgagttta tgagaatgtg
ggcggaattt gaatgggaaa 2760ataaggtaac cgttaacaca cccctcacgg
aactttcaga atacctcgaa catctactca 2820aaagcacaaa tttgaaatgt
ttaacatcag aaaaagctct gagcgggcag tgtggtttta 2880tggcagccaa
tttatatgca aaatccattt ttggagaaga cgctttggcc aacttaagta
2940tagagaaacc ttttaataaa cccgatgcgc cagtaagcgg tcatattaga
ataagggcca 3000aaagtcaggg catggcctta agtttaggag acaaagtcaa
tatgacacag aagagcacac 3060aacataaagt agtagctgca taaataaaaa
cgtttttttc tgtttcaacc cttttcttca 3120ttttaaatca tattctgtaa
tttaattaat ttaatatcat attactggtt acacattgtt 3180agattataaa
tatcttttaa agaataaaat ttatttagct tttacaaaga gcggaaagca
3240atctttatct tcttctaata agatcaacat caagttttct ggaaaatata
atatataatt 3300taaagatgtt tagcatttat tagtgaaata catggcttac
tattttgatg acaaagtcag 3360tgtaatatag aaaagcagat agtagaaaat ag
33922955PRTDiabrotica virgifera 2Met Thr Ala Val Glu Gln Pro Cys
Tyr Thr Leu Ile Asn Leu Pro Thr 1 5 10 15 Asp Ser Glu Pro Tyr Asn
Glu Met Gln Leu Lys Met Asp Leu Glu Lys 20 25 30 Gly Glu Val Lys
Val Lys Ile Arg Ala Leu Glu Lys Ile Ile His Met 35 40 45 Ile Leu
Ala Gly Glu Arg Leu Pro Asn Gly Phe Leu Met Thr Ile Ile 50 55 60
Arg Asn Val Leu Pro Leu Gln Asp His Leu Ala Lys Lys Leu Leu Leu 65
70 75 80 Ile Phe Trp Glu Ile Val Pro Lys Thr Asn Pro Glu Gly Lys
Leu Leu 85 90 95 Gln Glu Met Ile Leu Val Cys Asp Ala Tyr Arg Lys
Asp Leu Gln His 100 105 110 Pro Asn Glu Phe Leu Arg Gly Ser Thr Leu
Arg Phe Leu Cys Lys Leu 115 120 125 Lys Glu Pro Glu Leu Leu Glu Pro
Leu Met Pro Ser Ile Arg Ala Cys 130 135 140 Leu Asp His Arg His Ser
Tyr Val Arg Arg Asn Ala Val Leu Ala Ile 145 150 155 160 Phe Thr Ile
Tyr Lys Asn Phe Glu Ala Leu Ile Pro Asp Ala Pro Glu 165 170 175 Leu
Ile Ser Asn Tyr Leu Asp Gly Glu Gln Asp Met Ser Cys Lys Arg 180 185
190 Asn Ala Phe Leu Met Leu Leu His Ala Asp Gln Glu Arg Ala Leu Ser
195 200 205 Tyr Leu Ala Ser Cys Leu Asp Gln Val Asn Ser Phe Gly Asp
Ile Leu 210 215 220 Gln Leu Val Ile Val Glu Leu Ile Tyr Lys Val Cys
His Ser Asn Pro 225 230 235 240 Ala Glu Arg Ser Arg Phe Ile Arg Cys
Ile Tyr Asn Leu Leu Asn Ser 245 250 255 Ser Ser Pro Ala Val Arg Tyr
Glu Ala Ala Gly Thr Leu Val Thr Leu 260 265 270 Ser Ser Ala Pro Thr
Ala Val Lys Ala Ala Ala Ser Cys Tyr Ile Glu 275 280 285 Leu Ile Ile
Lys Glu Ser Asp Asn Asn Val Lys Leu Ile Val Leu Asp 290 295 300 Arg
Leu Ile Ala Leu Lys Glu Leu Pro Asn His Glu Arg Ile Leu Gln 305 310
315 320 Asp Leu Val Met Asp Ile Leu Arg Val Leu Ser Ala Pro Asp Leu
Glu 325 330 335 Val Arg Lys Lys Thr Leu Ser Leu Ala Leu Glu Leu Val
Ser Ser Arg 340 345 350 Asn Ile Glu Glu Met Val Leu Val Leu Thr Lys
Glu Val Ser Lys Thr 355 360 365 Val Asp Ser Glu His Glu Asp Thr Gly
Lys Tyr Arg Gln Leu Leu Val 370 375 380 Arg Thr Leu His Ser Cys Ser
Ile Lys Phe Pro Asp Ile Ala Arg Ser 385 390 395 400 Val Ile Pro Val
Leu Ile Glu Phe Leu Ser Asp Asn Asn Glu Leu Ala 405 410 415 Ala Thr
Asp Val Leu Leu Phe Leu Arg Glu Ala Ile Gln Lys Phe Lys 420 425 430
Glu Leu Gln Pro Leu Ile Ile Glu Lys Leu Ile Glu Thr Phe Lys Asp 435
440 445 Ile Lys Leu Val Lys Val His Arg Ala Ala Ile Trp Ile Leu Gly
Glu 450 455 460 Tyr Ala Ser Thr Ala Ser Asp Ile Glu Val Ile Val Gly
Glu Ile Asn 465 470 475 480 Arg Leu Leu Gly Glu Gly Ser Leu Val Glu
Ala Glu Gln Lys Leu Ile 485 490 495 Ala Gly Asp Thr Glu Glu Asn Ala
Pro Ala Pro Ala Ala Gly Ala Thr 500 505 510 Thr Leu Val Thr Ser Asp
Gly Thr Tyr Ala Thr Gln Ser Ala Phe Asn 515 520 525 Thr Val Ser Gln
Thr Thr Lys Glu Ala Arg Pro Pro Leu Arg Gln Tyr 530 535 540 Leu Met
Asp Gly Asp Phe Phe Ile Gly Ala Ser Leu Ala Ser Thr Leu 545 550 555
560 Thr Lys Leu Ser Leu Arg Tyr Glu Asp Leu Thr Ser Pro Ala Ala Ser
565 570 575 Asn Gly Phe Asn Ala Lys Ile Met Leu Ile Met Ala Gly Ile
Leu His 580 585 590 Leu Gly Lys Ser Gly Leu Pro Thr Lys Ser Ile Thr
Asn Asp Asp Lys 595 600 605 Asp His Ile Leu Phe Cys Leu Arg Val Leu
Ser Asp Arg Ser Pro Ile 610 615 620 Ile Val Glu Ile Phe Lys Lys Leu
Cys Arg Ser Ala Leu Asn Glu Met 625 630 635 640 Leu Leu Ala Lys Glu
Ser Val Glu Ala Ile Ser Gln Lys Ser Lys Glu 645 650 655 Lys Asn Lys
Arg Thr Ile Gln Thr Asp Asp Ala Ile Ser Phe Leu Gln 660 665 670 Leu
Glu Thr Asp Lys Ser Gly Glu Leu Gly Glu Asn Val Phe Glu Met 675 680
685 Ser Leu Ser Gln Ala Leu Val Gly Gly Arg Thr Gly Gly Gly Glu Ser
690 695 700 Val Leu Ser Ser Asn Lys Leu Asp Lys Ile Thr Gln Leu Thr
Gly Phe 705 710 715 720 Ser Asp Pro Val Tyr Ser Glu Ala Tyr Val His
Val Asn Gln Tyr Asp 725 730 735 Ile Val Leu Asp Val Leu Ile Val Asn
Gln Thr Asn Asp Thr Leu Gln 740 745 750 Asn Cys Thr Leu Glu Leu Ala
Thr Leu Gly Asp Leu Lys Leu Val Glu 755 760 765 Lys Pro Gln Pro Val
Val Leu Ala Pro Lys Asp Phe Cys Asn Ile Lys 770 775 780 Ala Asn Val
Lys Val Ala Ser Thr Glu Asn Gly Ile Ile Phe Gly Asn 785 790 795 800
Ile Val Tyr Asp Val Ile Gly Ala Gly Ser Asp Arg Asn Val Val Val 805
810 815 Leu Asn Asp Ile His Ile Asp Ile Met Asp Tyr Ile Val Pro Ala
Ser 820 825 830 Cys Thr Asp Ser Glu Phe Met Arg Met Trp Ala Glu Phe
Glu Trp Glu 835 840 845 Asn Lys Val Thr Val Asn Thr Pro Leu Thr Glu
Leu Ser Glu Tyr Leu 850 855 860 Glu His Leu Leu Lys Ser Thr Asn Leu
Lys Cys Leu Thr Ser Glu Lys 865 870 875 880 Ala Leu Ser Gly Gln Cys
Gly Phe Met Ala Ala Asn Leu Tyr Ala Lys 885 890 895 Ser Ile Phe Gly
Glu Asp Ala Leu Ala Asn Leu Ser Ile Glu Lys Pro 900 905 910 Phe Asn
Lys Pro Asp Ala Pro Val Ser Gly His Ile Arg Ile Arg Ala 915 920 925
Lys Ser Gln Gly Met Ala Leu Ser Leu Gly Asp Lys Val Asn Met Thr 930
935 940 Gln Lys Ser Thr Gln His Lys Val Val Ala Ala 945 950 955
3667DNADiabrotica virgifera 3gttagtaagg actctacatt cgtgttccat
taagttccca gatatcgcac gtagtgttat 60accagtcttg attgaatttt tatccgataa
taatgaactg gctgccacag atgtattgct 120gttcttaagg gaagccatac
agaagtttaa agaattgcaa ccgttaatta ttgagaaact 180catcgaaact
ttcaaagaca ttaaattggt caaagtccat agagcagcaa tttggatttt
240gggagaatac gcgagtactg cttccgatat agaagttatc gttggagaaa
ttaacagatt 300gttgggtgaa ggatccctcg ttgaagctga gcagaagtta
atagcaggag atacggaaga 360gaatgctcct gcacctgctg caggcgccac
cactttagtt acttccgatg gaacatatgc 420tacccaatca gctttcaaca
ctgtcagcca aaccactaaa gaagcacgac ctcctctaag 480acaatacctc
atggatggtg attttttcat cggagcctct ttggcatcta cattaaccaa
540actgtctttg cggtatgagg acctcacctc tcctgctgct agcaatggat
tcaatgccaa 600aattatgctt attatggctg gaattcttca cttgggaaaa
tcaggacttc ccacaaaatc 660aataacc 667424DNAArtificial
Sequencesynthesized promotor oligonucleotide 4ttaatacgac tcactatagg
gaga 245503DNAArtificial Sequencesynthesized partial coding region
5caccatgggc tccagcggcg ccctgctgtt ccacggcaag atcccctacg tggtggagat
60ggagggcaat gtggatggcc acaccttcag catccgcggc aagggctacg gcgatgccag
120cgtgggcaag gtggatgccc agttcatctg caccaccggc gatgtgcccg
tgccctggag 180caccctggtg accaccctga cctacggcgc ccagtgcttc
gccaagtacg gccccgagct 240gaaggatttc tacaagagct gcatgcccga
tggctacgtg caggagcgca ccatcacctt 300cgagggcgat ggcaatttca
agacccgcgc cgaggtgacc ttcgagaatg gcagcgtgta 360caatcgcgtg
aagctgaatg gccagggctt caagaaggat ggccacgtgc tgggcaagaa
420tctggagttc aatttcaccc cccactgcct gtacatctgg ggcgatcagg
ccaatcacgg 480cctgaagagc gccttcaaga tct 503652DNAArtificial
Sequencesynthesized primer oligonucleotide 6ttaatacgac tcactatagg
gagagttagt aaggactcta cattcgtgtt cc 52748DNAArtificial
Sequencesynthesized primer oligonucleotide 7ttaatacgac tcactatagg
gagaggttat tgattttgtg ggaagtcc 488425DNAArtificial
Sequencesynthesized artificial sequence 8attttgggag aatacgcgag
tactgcttcc gatatagaag ttatcgttgg agaaattaac 60agattgttgg gtgaaggatc
cctcgttgaa gctgagcaga gactagtacc ggttgggaaa 120ggtatgtttc
tgcttctacc tttgatatat atataataat tatcactaat tagtagtaat
180atagtatttc aagtattttt ttcaaaataa aagaatgtag tatatagcta
ttgcttttct 240gtagtttata agtgtgtata ttttaattta taacttttct
aatatatgac caaaacatgg 300tgatgtgcag gttgatccgc ggttatctgc
tcagcttcaa cgagggatcc ttcacccaac 360aatctgttaa tttctccaac
gataacttct atatcggaag cagtactcgc gtattctccc 420aaaat
4259471DNAArtificial Sequencesynthesized artificial sequence
9atgtcatctg gagcacttct ctttcatggg aagattcctt acgttgtgga gatggaaggg
60aatgttgatg gccacacctt tagcatacgt gggaaaggct acggagatgc ctcagtggga
120aaggactagt accggttggg aaaggtatgt ttctgcttct acctttgata
tatatataat 180aattatcact aattagtagt aatatagtat ttcaagtatt
tttttcaaaa taaaagaatg 240tagtatatag ctattgcttt tctgtagttt
ataagtgtgt atattttaat ttataacttt 300tctaatatat gaccaaaaca
tggtgatgtg caggttgatc cgcggttact ttcccactga 360ggcatctccg
tagcctttcc cacgtatgct aaaggtgtgg ccatcaacat tcccttccat
420ctccacaacg taaggaatct tcccatgaaa gagaagtgct ccagatgaca t
47110225DNASolanum tuberosum 10gactagtacc ggttgggaaa ggtatgtttc
tgcttctacc tttgatatat atataataat 60tatcactaat tagtagtaat atagtatttc
aagtattttt ttcaaaataa aagaatgtag 120tatatagcta ttgcttttct
gtagtttata agtgtgtata ttttaattta taacttttct 180aatatatgac
caaaacatgg tgatgtgcag gttgatccgc ggtta 22511705DNAArtificial
Sequencesynthesized artificial sequence 11atgtcatctg gagcacttct
ctttcatggg aagattcctt acgttgtgga gatggaaggg 60aatgttgatg gccacacctt
tagcatacgt gggaaaggct acggagatgc ctcagtggga 120aaggttgatg
cacagttcat ctgcacaact ggtgatgttc ctgtgccttg gagcacactt
180gtcaccactc tcacctatgg agcacagtgc tttgccaagt atggtccaga
gttgaaggac 240ttctacaagt cctgtatgcc agatggctat gtgcaagagc
gcacaatcac ctttgaagga 300gatggcaact tcaagactag ggctgaagtc
acctttgaga atgggtctgt ctacaatagg 360gtcaaactca atggtcaagg
cttcaagaaa gatggtcatg tgttgggaaa gaacttggag 420ttcaacttca
ctccccactg cctctacatc tggggtgacc aagccaacca cggtctcaag
480tcagccttca agatctgtca tgagattact ggcagcaaag gcgacttcat
agtggctgac 540cacacccaga tgaacactcc cattggtgga ggtccagttc
atgttccaga gtatcatcac 600atgtcttacc atgtgaaact ttccaaagat
gtgacagacc acagagacaa catgtccttg 660aaagaaactg tcagagctgt
tgactgtcgc aagacctacc tttga 70512218DNADiabrotica virgifera
12tagctctgat gacagagccc atcgagtttc aagccaaaca gttgcataaa gctatcagcg
60gattgggaac tgatgaaagt acaatmgtmg aaattttaag tgtmcacaac aacgatgaga
120ttataagaat ttcccaggcc tatgaaggat tgtaccaacg mtcattggaa
tctgatatca 180aaggagatac ctcaggaaca ttaaaaaaga attattag
21813424DNADiabrotica virgiferamisc_feature(393)..(393)n is a, c,
g, or tmisc_feature(394)..(394)n is a, c, g, or
tmisc_feature(395)..(395)n is a, c, g, or t 13ttgttacaag ctggagaact
tctctttgct ggaaccgaag agtcagtatt taatgctgta 60ttctgtcaaa gaaataaacc
acaattgaat ttgatattcg acaaatatga agaaattgtt 120gggcatccca
ttgaaaaagc cattgaaaac gagttttcag gaaatgctaa acaagccatg
180ttacacctta tccagagcgt aagagatcaa gttgcatatt tggtaaccag
gctgcatgat 240tcaatggcag gcgtcggtac tgacgataga actttaatca
gaattgttgt ttcgagatct 300gaaatcgatc tagaggaaat caaacaatgc
tatgaagaaa tctacagtaa aaccttggct 360gataggatag cggatgacac
atctggcgac tannnaaaag ccttattagc cgttgttggt 420taag
42414397DNADiabrotica virgifera 14agatgttggc tgcatctaga gaattacaca
agttcttcca tgattgcaag gatgtactga 60gcagaatagt ggaaaaacag gtatccatgt
ctgatgaatt gggaagggac gcaggagctg 120tcaatgccct tcaacgcaaa
caccagaact tcctccaaga cctacaaaca ctccaatcga 180acgtccaaca
aatccaagaa gaatcagcta aacttcaagc tagctatgcc ggtgatagag
240ctaaagaaat caccaacagg gagcaggaag tggtagcagc ctgggcagcc
ttgcagatcg 300cttgcgatca gagacacgga aaattgagcg atactggtga
tctattcaaa ttctttaact 360tggtacgaac gttgatgcag tggatggacg aatggac
39715490DNADiabrotica virgifera 15gcagatgaac accagcgaga aaccaagaga
tgttagtggt gttgaattgt tgatgaacaa 60ccatcagaca ctcaaggctg agatcgaagc
cagagaagac aactttacgg cttgtatttc 120tttaggaaag gaattgttga
gccgtaatca ctatgctagt gctgatatta aggataaatt 180ggtcgcgttg
acgaatcaaa ggaatgctgt actacagagg tgggaagaaa gatgggagaa
240cttgcaactc atcctcgagg tataccaatt cgccagagat gcggccgtcg
ccgaagcatg 300gttgatcgca caagaacctt acttgatgag ccaagaacta
ggacacacca ttgacgacgt 360tgaaaacttg ataaagaaac acgaagcgtt
cgaaaaatcg gcagcggcgc aagaagagag 420attcagtgct ttggagagac
tgacgacgtt cgaattgaga gaaataaaga ggaaacaaga 480agctgcccag
49016330DNADiabrotica virgifera 16agtgaaatgt tagcaaatat aacatccaag
tttcgtaatt gtacttgctc agttagaaaa 60tattctgtag tttcactatc ttcaaccgaa
aatagaataa atgtagaacc tcgcgaactt 120gcctttcctc caaaatatca
agaacctcga caagtttggt tggagagttt agatacgata 180gacgacaaaa
aattgggtat tcttgagctg catcctgatg tttttgctac taatccaaga
240atagatatta tacatcaaaa tgttagatgg caaagtttat atagatatgt
aagctatgct 300catacaaagt caagatttga agtgagaggt
33017320DNADiabrotica virgifera 17caaagtcaag atttgaagtg agaggtggag
gtcgaaaacc gtggccgcaa aagggattgg 60gacgtgctcg acatggttca attagaagtc
cactttggag aggtggagga gttgttcatg 120gaccaaaatc tccaacccct
catttttaca tgattccatt ctacacccgt ttgctgggtt 180tgactagcgc
actttcagta aaatttgccc aagatgactt gcacgttgtg gatagtctag
240atctgccaac tgacgaacaa agttatatag aagagctggt caaaagccgc
ttttgggggt 300ccttcttgtt ttatttgtag 3201847DNAArtificial
SequencePrimer oligonucleotide 18ttaatacgac tcactatagg gagacaccat
gggctccagc ggcgccc 471923DNAArtificial Sequencesynthesized primer
oligonucleotide 19agatcttgaa ggcgctcttc agg 232023DNAArtificial
Sequencesynthesized primer oligonucleotide 20caccatgggc tccagcggcg
ccc 232147DNAArtificial Sequencesynthesized primer oligonucleotide
21ttaatacgac tcactatagg gagaagatct tgaaggcgct cttcagg
472246DNAArtificial Sequencesynthesized primer oligonucleotide
22ttaatacgac tcactatagg gagagctcca acagtggttc cttatc
462329DNAArtificial Sequencesynthesized primer oligonucleotide
23ctaataattc ttttttaatg ttcctgagg 292422DNAArtificial
Sequencesynthesized primer oligonucleotide 24gctccaacag tggttcctta
tc 222553DNAArtificial Sequencesynthesized primer oligonucleotide
25ttaatacgac tcactatagg gagactaata attctttttt aatgttcctg agg
532648DNAArtificial Sequencesynthesized primer oligonucleotide
26ttaatacgac tcactatagg gagattgtta caagctggag aacttctc
482724DNAArtificial Sequencesynthesized primer oligonucleotide
27cttaaccaac aacggctaat aagg 242824DNAArtificial
Sequencesynthesized primer oligonucleotide 28ttgttacaag ctggagaact
tctc 242948DNAArtificial Sequencesynthesized primer oligonucleotide
29ttaatacgac tcactatagg gagacttaac caacaacggc taataagg
483047DNAArtificial Sequencesynthesized primer oligonucleotide
30ttaatacgac tcactatagg gagaagatgt tggctgcatc tagagaa
473122DNAArtificial Sequencesynthesized primer oligonucleotide
31gtccattcgt ccatccactg ca 223223DNAArtificial Sequencesynthesized
primer oligonucleotide 32agatgttggc tgcatctaga gaa
233346DNAArtificial Sequencesynthesized primer oligonucleotide
33ttaatacgac tcactatagg gagagtccat tcgtccatcc actgca
463446DNAArtificial Sequencesynthesized primer oligonucleotide
34ttaatacgac tcactatagg gagagcagat gaacaccagc gagaaa
463522DNAArtificial Sequencesynthesized primer oligonucleotide
35ctgggcagct tcttgtttcc tc 223622DNAArtificial Sequencesynthesized
primer oligonucleotide 36gcagatgaac accagcgaga aa
223746DNAArtificial Sequencesynthesized primer oligonucleotide
37ttaatacgac tcactatagg gagactgggc agcttcttgt ttcctc
463851DNAArtificial Sequencesynthesized primer oligonucleotide
38ttaatacgac tcactatagg gagaagtgaa atgttagcaa atataacatc c
513926DNAArtificial Sequencesynthesized primer oligonucleotide
39acctctcact tcaaatcttg actttg 264027DNAArtificial
Sequencesynthesized primer oligonucleotide 40agtgaaatgt tagcaaatat
aacatcc 274150DNAArtificial Sequencesynthesized primer
oligonucleotide 41ttaatacgac tcactatagg gagaacctct cacttcaaat
cttgactttg 504250DNAArtificial Sequencesynthesized primer
oligonucleotide 42ttaatacgac tcactatagg gagacaaagt caagatttga
agtgagaggt 504325DNAArtificial Sequencesynthesized primer
oligonucleotide 43ctacaaataa aacaagaagg acccc 254426DNAArtificial
Sequencesynthesized primer oligonucleotide 44caaagtcaag atttgaagtg
agaggt 264549DNAArtificial Sequencesynthesized primer
oligonucleotide 45ttaatacgac tcactatagg gagactacaa ataaaacaag
aaggacccc 49461150DNAZea mays 46caacggggca gcactgcact gcactgcaac
tgcgaatttc cgtcagcttg gagcggtcca 60agcgccctgc gaagcaaact acgccgatgg
cttcggcggc ggcgtgggag ggtccgacgg 120ccgcggagct gaagacagcg
ggggcggagg tgattcccgg cggcgtgcga gtgaaggggt 180gggtcatcca
gtcccacaaa ggccctatcc tcaacgccgc ctctctgcaa cgctttgaag
240atgaacttca aacaacacat ttacctgaga tggtttttgg agagagtttc
ttgtcacttc 300aacatacaca aactggcatc aaatttcatt ttaatgcgct
tgatgcactc aaggcatgga 360agaaagaggc actgccacct gttgaggttc
ctgctgcagc aaaatggaag ttcagaagta 420agccttctga ccaggttata
cttgactacg actatacatt tacgacacca tattgtggga 480gtgatgctgt
ggttgtgaac tctggcactc cacaaacaag tttagatgga tgcggcactt
540tgtgttggga ggatactaat gatcggattg acattgttgc cctttcagca
aaagaaccca 600ttcttttcta cgacgaggtt atcttgtatg aagatgagtt
agctgacaat ggtatctcat 660ttcttactgt gcgagtgagg gtaatgccaa
ctggttggtt tctgcttttg cgtttttggc 720ttagagttga tggtgtactg
atgaggttga gagacactcg gttacattgc ctgtttggaa 780acggcgacgg
agccaagcca gtggtacttc gtgagtgctg ctggagggaa gcaacatttg
840ctactttgtc tgcgaaagga tatccttcgg actctgcagc gtacgcggac
ccgaacctta 900ttgcccataa gcttcctatt gtgacgcaga agacccaaaa
gctgaaaaat cctacctgac 960tgacacaaag gcgccctacc gcgtgtacat
catgactgtc ctgtcctatc gttgcctttt 1020gtgtttgcca catgttgtgg
atgtacgttt ctatgacgaa acaccatagt ccatttcgcc 1080tgggccgaac
agagatagct gattgtcatg tcacgtttga attagaccat tccttagccc
1140tttttccccc 11504722DNAArtificial Sequencesynthesized primer
oligonucleotidemisc_feature(22)..(22)n is a, c, g, or t
47tttttttttt tttttttttt vn 224820DNAArtificial Sequencesynthesized
primer oligonucleotide 48ttgtgatgtt ggtggcgtat 204924DNAArtificial
Sequencesynthesized primer oligonucleotide 49tgttaaataa aaccccaaag
atcg 245021DNAArtificial Sequencesynthesized primer oligonucleotide
50tgagggtaat gccaactggt t 215124DNAArtificial Sequencesynthesized
primer oligonucleotide 51gcaatgtaac cgagtgtctc tcaa
245232DNAArtificial Sequencesynthesized probe oligonucleotide
52tttttggctt agagttgatg gtgtactgat ga 3253151DNAEscherichia coli
53gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc
60ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga
120cgacatcatt ccgtggcgtt atccagctaa g 1515469DNAArtificial
Sequencesynthesized partial coding region 54tgttcggttc cctctaccaa
gcacagaacc gtcgcttcag caacacctca gtcaaggtga 60tggatgttg
69554233DNAZea mays 55agcctggtgt ttccggagga gacagacatg atccctgccg
ttgctgatcc gacgacgctg 60gacggcgggg gcgcgcgcag gccgttgctc ccggagacgg
accctcgggg gcgtgctgcc 120gccggcgccg agcagaagcg gccgccggct
acgccgaccg ttctcaccgc cgtcgtctcc 180gccgtgctcc tgctcgtcct
cgtggcggtc acagtcctcg cgtcgcagca cgtcgacggg 240caggctgggg
gcgttcccgc gggcgaagat gccgtcgtcg tcgaggtggc cgcctcccgt
300ggcgtggctg agggcgtgtc ggagaagtcc acggccccgc tcctcggctc
cggcgcgctc 360caggacttct cctggaccaa cgcgatgctg gcgtggcagc
gcacggcgtt ccacttccag 420ccccccaaga actggatgaa cggttagttg
gacccgtcgc catcggtgac gacgcgcgga 480tcgttttttt cttttttcct
ctcgttctgg ctctaacttg gttccgcgtt tctgtcacgg 540acgcctcgtg
cacatggcga tacccgatcc gccggccgcg tatatctatc tacctcgacc
600ggcttctcca gatccgaacg gtaagttgtt ggctccgata cgatcgatca
catgtgagct 660cggcatgctg cttttctgcg cgtgcatgcg gctcctagca
ttccacgtcc acgggtcgtg 720acatcaatgc acgatataat cgtatcggta
cagagatatt gtcccatcag ctgctagctt 780tcgcgtattg atgtcgtgac
attttgcacg caggtccgct gtatcacaag ggctggtacc 840acctcttcta
ccagtggaac ccggactccg cggtatgggg caacatcacc tggggccacg
900ccgtctcgcg cgacctcctc cactggctgc acctaccgct ggccatggtg
cccgatcacc 960cgtacgacgc caacggcgtc tggtccgggt cggcgacgcg
cctgcccgac ggccggatcg 1020tcatgctcta cacgggctcc acggcggagt
cgtcggcgca ggtgcagaac ctcgcggagc 1080cggccgacgc gtccgacccg
ctgctgcggg agtgggtcaa gtcggacgcc aacccggtgc 1140tggtgccgcc
gccgggcatc gggccgacgg acttccgcga cccgacgacg gcgtgtcgga
1200cgccggccgg caacgacacg gcgtggcggg tcgccatcgg gtccaaggac
cgggaccacg 1260cggggctggc gctggtgtac cggacggagg acttcgtgcg
gtacgacccg gcgccggcgc 1320tgatgcacgc cgtgccgggc accggcatgt
gggagtgcgt ggacttctac ccggtggccg 1380cgggatcagg cgccgcggcg
ggcagcgggg acgggctgga gacgtccgcg gcgccgggac 1440ccggggtgaa
gcacgtgctc aaggctagcc tcgacgacga caagcacgac tactacgcga
1500tcggcaccta cgacccggcg acggacacct ggacccccga cagcgcggag
gacgacgtcg 1560ggatcggcct ccggtacgac tatggcaagt actacgcgtc
gaagaccttc tacgaccccg 1620tccttcgccg gcgggtgctc tgggggtggg
tcggcgagac cgacagcgag cgcgcggaca 1680tcctcaaggg ctgggcatcc
gtgcaggtac gtctcagggt ttgaggctag catggcttca 1740atcttgctgg
catcgaatca ttaatgggca gatattataa cttgataatc tgggttggtt
1800gtgtgtggtg gggatggtga cacacgcgcg gtaataatgt agctaagctg
gttaaggatg 1860agtaatgggg ttgcgtataa acgacagctc tgctaccatt
acttctgaca cccgattgaa 1920ggagacaaca gtaggggtag ccggtagggt
tcgtcgactt gccttttctt ttttcctttg 1980ttttgttgtg gatcgtccaa
cacaaggaaa ataggatcat ccaacaaaca tggaagtaat 2040cccgtaaaac
atttctcaag gaaccatcta gctagacgag cgtggcatga tccatgcatg
2100cacaaacact agataggtct ctgcagctgt gatgttcctt tacatatacc
accgtccaaa 2160ctgaatccgg tctgaaaatt gttcaagcag agaggccccg
atcctcacac ctgtacacgt 2220ccctgtacgc gccgtcgtgg tctcccgtga
tcctgccccg tcccctccac gcggccacgc 2280ctgctgcagc gctctgtaca
agcgtgcacc acgtgagaat ttccgtctac tcgagcctag 2340tagttagacg
ggaaaacgag aggaagcgca cggtccaagc acaacacttt gcgcgggccc
2400gtgacttgtc tccggttggc tgagggcgcg cgacagagat gtatggcgcc
gcggcgtgtc 2460ttgtgtcttg tcttgcctat acaccgtagt cagagactgt
gtcaaagccg tccaacgaca 2520atgagctagg aaacgggttg gagagctggg
ttcttgcctt gcctcctgtg atgtctttgc 2580cttgcatagg gggcgcagta
tgtagctttg cgttttactt cacgccaaag gatactgctg 2640atcgtgaatt
attattatta tatatatatc gaatatcgat ttcgtcgctc tcgtggggtt
2700ttattttcca gactcaaact tttcaaaagg cctgtgtttt agttcttttc
ttccaattga 2760gtaggcaagg cgtgtgagtg tgaccaacgc atgcatggat
atcgtggtag actggtagag 2820ctgtcgttac cagcgcgatg cttgtatatg
tttgcagtat tttcaaatga atgtctcagc 2880tagcgtacag ttgaccaagt
cgacgtggag ggcgcacaac agacctctga cattattcac 2940ttttttttta
ccatgccgtg cacgtgcagt caatccccag gacggtcctc ctggacacga
3000agacgggcag caacctgctc cagtggccgg tggtggaggt ggagaacctc
cggatgagcg 3060gcaagagctt cgacggcgtc gcgctggacc gcggatccgt
cgtgcccctc gacgtcggca 3120aggcgacgca ggtgacgccg cacgcagcct
gctgcagcga acgaactcgc gcgttgccgg 3180cccgcggcca gctgacttag
tttctctggc tgatcgaccg tgtgcctgcg tgcgtgcagt 3240tggacatcga
ggctgtgttc gaggtggacg cgtcggacgc ggcgggcgtc acggaggccg
3300acgtgacgtt caactgcagc accagcgcag gcgcggcggg ccggggcctg
ctcggcccgt 3360tcggccttct cgtgctggcg gacgacgact tgtccgagca
gaccgccgtg tacttctacc 3420tgctcaaggg cacggacggc agcctccaaa
ctttcttctg ccaagacgag ctcaggtatg 3480tatgttatga cttatgacca
tgcatgcatg cgcatttctt agctaggctg tgaagcttct 3540tgttgagttg
tttcacagat gcttaccgtc tgctttgttt cgtatttcga ctaggcatcc
3600aaggcgaacg atctggttaa gagagtatac gggagcttgg tccctgtgct
agatggggag 3660aatctctcgg tcagaatact ggtaagtttt tacagcgcca
gccatgcatg tgttggccag 3720ccagctgctg gtactttgga cactcgttct
tctcgcactg ctcattattg cttctgatct 3780ggatgcacta caaattgaag
gttgaccact ccatcgtgga gagctttgct caaggcggga 3840ggacgtgcat
cacgtcgcga gtgtacccca cacgagccat ctacgactcc gcccgcgtct
3900tcctcttcaa caacgccaca catgctcacg tcaaagcaaa atccgtcaag
atctggcagc 3960tcaactccgc ctacatccgg ccatatccgg caacgacgac
ttctctatga ctaaattaag 4020tgacggacag ataggcgata ttgcatactt
gcatcatgaa ctcatttgta caacagtgat 4080tgtttaattt atttgctgcc
ttccttatcc ttcttgtgaa actatatggt acacacatgt 4140atcattaggt
ctagtagtgt tgttgcaaag acacttagac accagaggtt ccaggagtat
4200cagagataag gtataagagg gagcagggag cag 42335620DNAArtificial
Sequencesynthesized primer oligonucleotide 56tgttcggttc cctctaccaa
205722DNAArtificial Sequencesynthesized primer oligonucleotide
57caacatccat caccttgact ga 225824DNAArtificial Sequencesynthesized
probe oligonucleotide 58cacagaaccg tcgcttcagc aaca
245918DNAArtificial Sequencesynthesized primer oligonucleotide
59tggcggacga cgacttgt 186019DNAArtificial Sequencesynthesized
primer oligonucleotide 60aaagtttgga ggctgccgt 196126DNAArtificial
Sequencesynthesized probe oligonucleotide 61cgagcagacc gccgtgtact
tctacc 266219DNAArtificial Sequencesynthesized primer
oligonucleotide 62cttagctgga taacgccac 196319DNAArtificial
Sequencesynthesized primer oligonucleotide 63gaccgtaagg cttgatgaa
196421DNAArtificial Sequencesynthesized probe oligonucleotide
64cgagattctc cgcgctgtag a 216525DNAArtificial Sequencesynthesized
primer oligonucleotide 65gtatgtttct gcttctacct ttgat
256629DNAArtificial Sequencesynthesized primer oligonucleotide
66ccatgttttg gtcatatatt agaaaagtt 296734DNAArtificial
Sequencesynthesized probe oligonucleotide 67agtaatatag tatttcaagt
atttttttca aaat 3468384DNADiabrotica virgifera 68gatgtcgctg
tcacaagctt tagtaggagg tcgaacggga ggtggcgaat cagtattaag 60ttccaataaa
ttagataaaa tcacacaact gactggtttt tccgatccag tttattccga
120agcatacgtt cacgtgaatc agtacgatat cgtgcttgat gtcttaatcg
taaaccaaac 180taacgatact ttacaaaact gcacgctaga gctggctact
ttaggcgatt tgaagttggt 240agagaagcca caacctgtcg tattggcgcc
caaagacttt tgcaacatta aagctaacgt 300gaaagtggcc tcaactgaaa
acggaattat atttggcaac attgtgtatg atgtcatagg 360agcggggtca
gataggaatg ttgt 38469390DNADiabrotica virgifera 69ggactatata
gtgcctgcta gttgtacaga tagcgagttt atgagaatgt gggcggaatt 60tgaatgggaa
aataaggtaa ccgttaacac acccctcacg gaactttcag aatacctcga
120acatctactc aaaagcacaa atttgaaatg tttaacatca gaaaaagctc
tgagcgggca 180gtgtggtttt atggcagcca atttatatgc aaaatccatt
tttggagaag acgctttggc 240caacttaagt atagagaaac cttttaataa
acccgatgcg ccagtaagcg gtcatattag 300aataagggcc aaaagtcagg
gcatggcctt aagtttagga gacaaagtca atatgacaca 360gaagagcaca
caacataaag tagtagctgc 39070122DNADiabrotica virgifera 70gaagagaatg
ctcctgcacc tgctgcaggc gccaccactt tagttacttc cgatggaaca 60tatgctaccc
aatcagcttt caacactgtc agccaaacca ctaaagaagc acgacctcct 120ct
12271100DNADiabrotica virgifera 71attttgggag aatacgcgag tactgcttcc
gatatagaag ttatcgttgg agaaattaac 60agattgttgg gtgaaggatc cctcgttgaa
gctgagcaga 10072524DNADiabrotica virgifera 72agccatcgct gattacctca
gcagtagtgg atacacggat gccttggagg cgttcaagaa 60ggaggcggat atgcccggcg
aggtggagag gaaattcggc ggcctgttgg agaaaaaatg 120gacgtccgtc
ataaggttgc aaaagaaagt aatggagtta gagtcaaaat tgaacgaagc
180cgaaaaagaa tacatagagg gcgctcccac gcgaaacaaa cgtacccctt
cggaatggat 240accgcggccg cctgaaaagt tctgtctcac aggtcatcga
gcgcctgtga ctcgagtaat 300ctttcatcca gtcttcagtc tcatggtttc
ggcaagtgaa gacgccacca tcaaagtgtg 360ggacttcgag actggtgaat
tcgaacgcac ccttaaaggt cacacggatt gcgtccagga 420tatagctttt
gatgcatcgg gtaaattact ggtatcctgt agtgccgaca tgagcatcaa
480actatgggat ttccaacaga cgtttgagtg cgtgcgcact atgt
5247348DNAArtificial Sequencesynthesized primer oligonucleotide
73ttaatacgac tcactatagg gagagatgtc gctgtcacaa gctttagt
487446DNAArtificial Sequencesynthesized primer oligonucleotide
74ttaatacgac tcactatagg gagaacaaca ttcctatctg accccg
467552DNAArtificial Sequencesynthesized primer oligonucleotide
75ttaatacgac tcactatagg gagaggacta tatagtgcct gctagttgta ca
527650DNAArtificial Sequencesynthesized primer oligonucleotide
76ttaatacgac tcactatagg gagagcagct actactttat gttgtgtgct
507744DNAArtificial Sequencesynthesized primer oligonucleotide
77ttaatacgac tcactatagg gagagaagag aatgctcctg cacc
447846DNAArtificial Sequencesynthesized primer oligonucleotide
78ttaatacgac tcactatagg gagaagagga ggtcgtgctt ctttag
467944DNAArtificial Sequencesynthesized primer oligonucleotide
79ttaatacgac tcactatagg gagaattttg ggagaatacg cgag
448043DNAArtificial Sequencesynthesized primer oligonucleotide
80ttaatacgac tcactatagg gagatctgct cagcttcaac gag
438144DNAArtificial Sequencesynthesized primer oligonucleotide
81ttaatacgac tcactatagg gagaagccat cgctgattac ctca
448244DNAArtificial Sequencesynthesized primer oligonucleotide
82ttaatacgac tcactatagg gagaacatag tgcgcacgca ctca
44833032DNAEuschistus heros 83attttttaaa attattttta aaaataaatt
acaaagtttc cataaacaaa acaatataat 60atattgtttt ttggataaga aacataacct
gacagccacg taactacggt tgagtggagg 120tttaagtgaa ttcatagtca
agttttagct tactgtaata atgcctctta gactggacat 180caaaaggaag
ctgacagcca gatctgatcg agttaagtgt gttgacttac atccatcaga
240accatggatg ttggcatctt tatacaatgg taatgttcac atttggaacc
atgagactca 300acagttagtg aaatcatttg aagtatgtga ccaaccagtt
cgtgctgcag tattcgtccc 360tcgtaagaat tggattgtaa caggatcaga
tgatatgcag atccgagttt ttaattacaa 420tactctcgaa agagtaaatg
catttgaagc acattcagac tacgtcagat gtatagcagt 480tcaccctgcc
catccttata tcttgacatc atcagatgat atgttaatta aactttggaa
540ttggtctaag gcatgggttt gccaacagat atttgaagga catacccact
atgttatgca 600agtggttata aatccaaaag ataataatac ttttgcatct
gcttcattag atcgtacagt 660gaaggtttgg cagttgggct cagctgctcc
aaattttact ttagagggtc atgaaaaagg 720agtcaattct gttgattatt
accatggagg cgacaaacct tatcttatat ctggtgctga 780tgatcatctt
gtaaaaatat gggattacca aaataagact tgtgttcaaa ccctggaggg
840gcatgcccaa aatattactg cagtttgttt tcacactgaa ctacccatta
taataactgg 900gtcagaagat ggtactgttc gattgtggca ctcagcaaca
taccgattag aatcatctct 960taactatggt ttagaacgtg tttggactat
tgctagactg aagggctcaa acaatatagc 1020tcttggttac gatgaaggga
gtattatggt aaagatagga cgagaagaac cagcaatttc 1080aatggatgtt
aatggtgaaa aaatagtctg ggccagacat tctgaaattg aacaagtcaa
1140cttgaaacaa gttacaggag aagtgagaga tggtgaacgt cttgctttag
ctcctaaaga 1200aatgggacca tgtgaaatat atcctcaaag catttcacat
aatccgaatg gaaggtttgt 1260tgttgtttgt ggagatggtg aatatataat
ttatacagcc atggctttaa gaaacaaaag 1320ttttggatca gctcaagagt
ttgtttgggc acaagacagt tctgattatg ctataagaga 1380aggaacttct
actgtaaaac tattcagaca gttcaaagag cgcaagacac ttaaaccaga
1440atttggtgca gaaggtattt ttggtggaca attacttggt gtcagatcag
tctcaggatt 1500atgtttatat gattgggaaa ctctggaatt agtgagaaga
atagaaattc aggctaaatc 1560tctacactgg tctgattccg gccatctcct
tgccattgtg actgacgatt catattatat 1620attgaagtat gattcatctg
cacttacaag tgctcaagag aggactccag atggtgtaga 1680agcagcgttt
actcttgttg gagaagttaa tgatacagtg aagactggat tgtgggttgg
1740tgattgtttt atttatacca atgctgtcgg acggattaat tactatgttg
gaggagaaat 1800agtaactgtt gctcacttgg attgcacaat gtacctgtta
ggatatgttg ccagacaaaa 1860tctcttatat ctttccgata aacaccataa
cattgtttgt tatacactat tactgtctgt 1920tcttgaatat caaaccgctg
ttatgagagg agattttgaa acagctgacc gtgtcttgcc 1980aacaattcca
gttcagcatc gttcacgagt tgctcatttc ttagaaaaac agggtttcaa
2040aaaagaagct ctcgctgtat ctactgatcc tgaacataaa tttgaactag
ctcttgggct 2100taaagagtta gatactgttg tccaattagc tgaagaaata
ggcagtactg tcaaatgggg 2160tcaagctgct gaattagcaa caagacaagc
caggcttgat gttgcacaag cagcccttca 2220ccgagcccag cattatggtg
gacttctgct gctctctaca tcagcaggta accgagaaat 2280gatggaaaaa
cttgggaaga gttctggaga aaacggaaaa aataatgtaa gctttcttgc
2340ttattttctt cttggagacc ttcagaaatg tcttcaaatt cttattgaca
cagatcgcat 2400cccagaagct gccttttttg caaggacata tttgccaagt
gaagtacctc gagttgttgg 2460cctatggcga ggtttagcca aggcagggca
gagccttgca gatccttcac agtatcccaa 2520tctctttcca ggttatgcag
atgctttaaa aactgagcaa tatttagcaa aagaatcctc 2580tatgtctaaa
ccagcttcat attacaaaaa tatcaagcca aatcatgaaa gacagcctat
2640agaagaaatg aaagctgctg aagaggaagg tggattccat tatgttcccc
tgaaatttga 2700agatgattct caaaaagaaa aacaaactga tgataagagt
gctgaagtac aacagcctac 2760aacacatatc aaagaaacaa aagaagaatt
tgctaagcag ccgaaaacag ctgactatga 2820tgatttagat ctagaaatag
aggcaatgaa tcttgatgat attgatccaa gtgatttaaa 2880tttggatgaa
gatctcctca atgactaata tacttattta tatgcctttt taaaaagtat
2940tttaaatgtt ttgttgagca ttgacataag caaaagggat ataattaaat
taagaaaaac 3000ttgtacatat ttcaaataat cttagatcat tg
3032843380DNAEuschistus heros 84cggcaatctt tgtgttgaaa cgataagtgt
gattgggttc tatgtaattc ttattgtatt 60attggatata atataatata tattatattg
taaaaattga tatgaactaa aaacgtatac 120ttgaacctaa ctatttgtat
aactcaactg catattaact ggtaaaagat ttaagtataa 180ttcgttagtc
aaggatggct gtggtagaac aaccttgtta tactctaata aactttccat
240ctgatttaga gcctcccaat gaaatgcagc tgaaatctga tttagaaaat
ggagacacta 300aaactaaaat tgaagctttg aaaaatatta ttcatttaat
tgcaaatgga gagcgtctac 360ctggcttact tatgcatatt atacgttttg
ttttaccttc gcaagatcat accattaaaa 420aattgctgct catcttttgg
gaaatcgttc ctaaaactac tccagatggc aaacttctcc 480aggaaatgat
tttagtttgt gatgcttatc gaaaggactt acaacatcct aatgagtttg
540taaggggatc cacactacgt tttctttgta aactaaaaga acctgaatta
cttgaaccct 600taatgcctgc tataagagct tgcttggagc atagagtttc
ttacgttcgg agaaatgcag 660tacttgcaat atttactatt tataggaatt
ttgaattctt agcacctgat gccccagagc 720tcatagctaa tttcttagat
ggtgagcaag atatgtcatg taaaagaaat gcctttttga 780tgctcttaca
tgctgatcaa gaacgtgctt tatcctactt ggcttcgtgt cttgatcaag
840tgacttcttt tggtgatata cttcagttag ttattgttga attaatatat
aaggtttgcc 900atgcgaaccc ttctgaacgt tctcgattta ttcgttgcat
ttataattta ctcaattcta 960atagtcctgc tgttcgatac gaagctgctg
gaactttaat taccctttca aatgctccta 1020ctgcaataaa agctgctgct
tcttgttata ttgatttgat aataaaggaa agtgacaata 1080atgttaaatt
aattgtatta gaccgtctaa tatctttaaa agagattcct acgcatgaga
1140gggttcttca agatttagta atggatatat tacgtgtcct agccagtcct
gacatggaag 1200ttaagaaaaa agtcttaagt ttagcgttag atctcaccac
ttcaaggtgt gtagaagaaa 1260tggttttaat gttaaaaaaa gaagttgcta
aaacacacaa ccttacagaa catgaagatg 1320ctggaaaata tcgtcaactt
cttgtgagaa ctcttcactc ctgttgcatg aagttcccag 1380atgttgctgc
ttcagttata ccagttttaa tggaatttct ttctgatact agtgagctag
1440cttcgtatga tgttcttata tttgttcgag aagcaattca taagtttgat
tccttaagga 1500ctctgataat agagaaatta ttagaagcat ttcctaccat
aaaatctatg aaagttcatc 1560gagctgctct ttggatattg ggtgaatata
ctacttcagt tggagacatt aaagaagtaa 1620tgaaacaaat taaaaatgcc
cttggagaaa tgccacttgt tgatgatgaa atgaaaagag 1680cttctggagg
agagaaagtt gaggaagttg atcatcgtga tcaaatgaag cttgttacat
1740ctgatggaac atatgctaca caatcaattt ttaacaccat tctggccatt
aaaaaagagg 1800atcgacctcc acttagacag tacttgattg atggcgactt
ttttattggc gtatctgtgg 1860cctctactct tgtaaaatta gccttacgct
ttaaagagct tgttcagcaa gaaaatatgt 1920acaacagatt ttttgctgag
tgtatgctta tcatttcatc catagtacgt ctgggtaaat 1980ctgataaaag
tttattttat ttaggatatc cttcaaaaca gctcagctat gatgattatg
2040aacgaatgtt gctttgtcta aaggttcttt ctgaaaatag tgcacctatt
gttaaaatat 2100ttaacactga ttgtcgcaat gctcttgcta acatgttagt
tgctcaacag aacgaggaat 2160attcccttat caaggccaaa gaaaagtctg
tccatactat ccaagttgat gatccagtct 2220catttttaca attatcaaca
atgcgatcat ctgattatgg ttctgaaaat gtttttgagc 2280ttagtttaaa
tcaagctgtt gggggaccaa atacagctac aaacacatct gaacttccat
2340tttcagctag taaattgaat aaagttactc agttgactgg gttttctgac
ccagtttatg 2400ctgaagcata tgttcatgtc aaccagtatg acattgtgct
tgatgttttg atcgttaatc 2460aaacaggtga tacacttcaa aactgtacac
ttgaactggc aactcttggt gatttgaaac 2520ttgttgaaaa acctcaacct
tgtgtattag ctccttatga tttctgtaat attaaagcaa 2580acgtgaaagt
tgcatcaact gaaaatggaa tcatatttgg aaatattgtt tatgatatca
2640gtggtgctgc ttctgataga aatgttgttg ttcttaatga tatccatata
gatatcatgg 2700attatattgt tcctgccact tgttctgata cagaatttcg
ccagatgtgg gctgaatttg 2760aatgggaaaa caaagtgtct gttaatactt
atttggttga tcttcatgaa tatcttggcc 2820acttactgaa gagcactaat
atgaaatgtt taacaccaga aaaagctctc tgcggtcaat 2880gtggttttat
ggctgctaac atgtatgccc gctcaatttt tggagaagat gccctggcta
2940acttaagtat tgaaaaacct ttcaataaac ccaatgcacc tgttactgga
catatcagaa 3000tcagagctaa aagtcagggt atggcattga gcttaggaga
taaaattaat atgactcaga 3060agaaacccgt tatcatggct cagtgaagca
ttatggtttt aatattaaaa ttaatttcac 3120aattgttgaa agctttttaa
cttattggtt tataattata attatttttt gaactatgat 3180tttgtgtaat
atatgtattt aagttatcaa tttttactta actccaataa aaaaacaata
3240ttaattgata tgatttccta caaatatata tatatattta ttttttttaa
tcttattacg 3300taatttttat ataattgact ttttcgattc gaatttacag
aactatgatt tttttacagg 3360tcaacacttc ttctacaact
338085498DNAEuschistus heros 85gaagggctca aacaatatag ctcttggtta
cgatgaaggg agtattatgg taaagatagg 60acgagaagaa ccagcaattt caatggatgt
taatggtgaa aaaatagtct gggccagaca 120ttctgaaatt gaacaagtca
acttgaaaca agttacagga gaagtgagag atggtgaacg 180tcttgcttta
gctcctaaag aaatgggacc atgtgaaata tatcctcaaa gcatttcaca
240taatccgaat ggaaggtttg ttgttgtttg tggagatggt gaatatataa
tttatacagc 300catggcttta agaaacaaaa gttttggatc agctcaagag
tttgtttggg cacaagacag 360ttctgattat gctataagag aaggaacttc
tactgtaaaa ctattcagac agttcaaaga 420gcgcaagaca cttaaaccag
aatttggtgc agaaggtatt tttggtggac aattacttgg 480tgtcagatca gtctcagg
49886441DNAEuschistus heros 86gccatgcgaa cccttctgaa cgttctcgat
ttattcgttg catttataat ttactcaatt 60ctaatagtcc tgctgttcga tacgaagctg
ctggaacttt aattaccctt tcaaatgctc 120ctactgcaat aaaagctgct
gcttcttgtt atattgattt gataataaag gaaagtgaca 180ataatgttaa
attaattgta ttagaccgtc taatatcttt aaaagagatt cctacgcatg
240agagggttct tcaagattta gtaatggata tattacgtgt cctagccagt
cctgacatgg 300aagttaagaa aaaagtctta agtttagcgt tagatctcac
cacttcaagg tgtgtagaag 360aaatggtttt aatgttaaaa aaagaagttg
ctaaaacaca caaccttaca gaacatgaag 420atgctggaaa atatcgtcaa c
4418750DNAArtificial Sequencesynthesized primer oligonucleotide
87ttaatacgac tcactatagg gagagaaggg ctcaaacaat atagctcttg
508853DNAArtificial Sequencesynthesized primer oligonucleotide
88ttaatacgac tcactatagg gagacctgag actgatctga caccaagtaa ttg
538949DNAArtificial Sequencesynthesized primer oligonucleotide
89ttaatacgac tcactatagg gagagccatg cgaacccttc tgaacgttc
499049DNAArtificial Sequencesynthesized primer oligonucleotide
90ttaatacgac tcactatagg gagagttgac gatattttcc agcatcttc
4991301DNAArtificial Sequencesynthesized artificial sequence
91catctggagc acttctcttt catgggaaga ttccttacgt tgtggagatg gaagggaatg
60ttgatggcca cacctttagc atacgtggga aaggctacgg agatgcctca gtgggaaagg
120ttgatgcaca gttcatctgc acaactggtg atgttcctgt gccttggagc
acacttgtca 180ccactctcac ctatggagca cagtgctttg ccaagtatgg
tccagagttg aaggacttct 240acaagtcctg tatgccagat ggctatgtgc
aagagcgcac aatcaccttt gaaggagatg 300g 3019247DNAArtificial
Sequencesynthesized primer oligonucleotide 92ttaatacgac tcactatagg
gagagcatct ggagcacttc tctttca 479346DNAArtificial
Sequencesynthesized primer oligonucleotide 93ttaatacgac tcactatagg
gagaccatct ccttcaaagg tgattg 4694915PRTEuschistus heros 94Met Pro
Leu Arg Leu Asp Ile Lys Arg Lys Leu Thr Ala Arg Ser Asp 1 5 10 15
Arg Val Lys Cys Val Asp Leu His Pro Ser Glu Pro Trp Met Leu Ala 20
25 30 Ser Leu Tyr Asn Gly Asn Val His Ile Trp Asn His Glu Thr Gln
Gln 35 40 45 Leu Val Lys Ser Phe Glu Val Cys Asp Gln Pro Val Arg
Ala Ala Val 50 55 60 Phe Val Pro Arg Lys Asn Trp Ile Val Thr Gly
Ser Asp Asp Met Gln 65 70 75 80 Ile Arg Val Phe Asn Tyr Asn Thr Leu
Glu Arg Val Asn Ala Phe Glu 85 90 95 Ala His Ser Asp Tyr Val Arg
Cys Ile Ala Val His Pro Ala His Pro 100 105 110 Tyr Ile Leu Thr Ser
Ser Asp Asp Met Leu Ile Lys Leu Trp Asn Trp 115 120 125 Ser Lys Ala
Trp Val Cys Gln Gln Ile Phe Glu Gly His Thr His Tyr 130 135 140 Val
Met Gln Val Val Ile Asn Pro Lys Asp Asn Asn Thr Phe Ala Ser 145 150
155 160 Ala Ser Leu Asp Arg Thr Val Lys Val Trp Gln Leu Gly Ser Ala
Ala 165 170 175 Pro Asn Phe Thr Leu Glu Gly His Glu Lys Gly Val Asn
Ser Val Asp 180 185 190 Tyr Tyr His Gly Gly Asp Lys Pro Tyr Leu Ile
Ser Gly Ala Asp Asp 195 200 205 His Leu Val Lys Ile Trp Asp Tyr Gln
Asn Lys Thr Cys Val Gln Thr 210 215 220 Leu Glu Gly His Ala Gln Asn
Ile Thr Ala Val Cys Phe His Thr Glu 225 230 235 240 Leu Pro Ile Ile
Ile Thr Gly Ser Glu Asp Gly Thr Val Arg Leu Trp 245 250 255 His Ser
Ala Thr Tyr Arg Leu Glu Ser Ser Leu Asn Tyr Gly Leu Glu 260 265 270
Arg Val Trp Thr Ile Ala Arg Leu Lys Gly Ser Asn Asn Ile Ala Leu 275
280 285 Gly Tyr Asp Glu Gly Ser Ile Met Val Lys Ile Gly Arg Glu Glu
Pro 290 295 300 Ala Ile Ser Met Asp Val Asn Gly Glu Lys Ile Val Trp
Ala Arg His 305 310 315 320 Ser Glu Ile Glu Gln Val Asn Leu Lys Gln
Val Thr Gly Glu Val Arg 325 330 335 Asp Gly Glu Arg Leu Ala Leu Ala
Pro Lys Glu Met Gly Pro Cys Glu 340 345 350 Ile Tyr Pro Gln Ser Ile
Ser His Asn Pro Asn Gly Arg Phe Val Val 355 360 365 Val Cys Gly Asp
Gly Glu Tyr Ile Ile Tyr Thr Ala Met Ala Leu Arg 370 375 380 Asn Lys
Ser Phe Gly Ser Ala Gln Glu Phe Val Trp Ala Gln Asp Ser 385 390 395
400 Ser Asp Tyr Ala Ile Arg Glu Gly Thr Ser Thr Val Lys Leu Phe Arg
405 410 415 Gln Phe Lys Glu Arg Lys Thr Leu Lys Pro Glu Phe Gly Ala
Glu Gly 420 425 430 Ile Phe Gly Gly Gln Leu Leu Gly Val Arg Ser Val
Ser Gly Leu Cys 435 440 445 Leu Tyr Asp Trp Glu Thr Leu Glu Leu Val
Arg Arg Ile Glu Ile Gln 450 455 460 Ala Lys Ser Leu His Trp Ser Asp
Ser Gly His Leu Leu Ala Ile Val 465 470 475 480 Thr Asp Asp Ser Tyr
Tyr Ile Leu Lys Tyr Asp Ser Ser Ala Leu Thr 485 490 495 Ser Ala Gln
Glu Arg Thr Pro Asp Gly Val Glu Ala Ala Phe Thr Leu 500 505 510 Val
Gly Glu Val Asn Asp Thr Val Lys Thr Gly Leu Trp Val Gly Asp 515 520
525 Cys Phe Ile Tyr Thr Asn Ala Val Gly Arg Ile Asn Tyr Tyr Val Gly
530 535 540 Gly Glu Ile Val Thr Val Ala His Leu Asp Cys Thr Met Tyr
Leu Leu 545 550 555 560 Gly Tyr Val Ala Arg Gln Asn Leu Leu Tyr Leu
Ser Asp Lys His His 565 570 575 Asn Ile Val Cys Tyr Thr Leu Leu Leu
Ser Val Leu Glu Tyr Gln Thr 580 585 590 Ala Val Met Arg Gly Asp Phe
Glu Thr Ala Asp Arg Val Leu Pro Thr 595 600 605 Ile Pro Val Gln His
Arg Ser Arg Val Ala His Phe Leu Glu Lys Gln 610 615 620 Gly Phe Lys
Lys Glu Ala Leu Ala Val Ser Thr Asp Pro Glu His Lys 625 630 635 640
Phe Glu Leu Ala Leu Gly Leu Lys Glu Leu Asp Thr Val Val Gln Leu 645
650 655 Ala Glu Glu Ile Gly Ser Thr Val Lys Trp Gly Gln Ala Ala Glu
Leu 660 665 670 Ala Thr Arg Gln Ala Arg Leu Asp Val Ala Gln Ala Ala
Leu His Arg 675 680 685 Ala Gln His Tyr Gly Gly Leu Leu Leu Leu Ser
Thr Ser Ala Gly Asn 690 695 700 Arg Glu Met Met Glu Lys Leu Gly Lys
Ser Ser Gly Glu Asn Gly Lys 705 710 715 720 Asn Asn Val Ser Phe Leu
Ala Tyr Phe Leu Leu Gly Asp Leu Gln Lys 725 730 735 Cys Leu Gln Ile
Leu Ile Asp Thr Asp Arg Ile Pro Glu Ala Ala Phe 740 745 750 Phe Ala
Arg Thr Tyr Leu Pro Ser Glu Val Pro Arg Val Val Gly Leu 755 760 765
Trp Arg Gly Leu Ala Lys Ala Gly Gln Ser Leu Ala Asp Pro Ser Gln 770
775 780 Tyr Pro Asn Leu Phe Pro Gly Tyr Ala Asp Ala Leu Lys Thr Glu
Gln 785 790 795 800 Tyr Leu Ala Lys Glu Ser Ser Met Ser Lys Pro Ala
Ser Tyr Tyr Lys 805 810 815 Asn Ile Lys Pro Asn His Glu Arg Gln Pro
Ile Glu Glu Met Lys Ala 820 825 830 Ala Glu Glu Glu Gly Gly Phe His
Tyr Val Pro Leu Lys Phe Glu Asp 835 840 845 Asp Ser Gln Lys Glu Lys
Gln Thr Asp Asp Lys Ser Ala Glu Val Gln 850 855 860 Gln Pro Thr Thr
His Ile Lys Glu Thr Lys Glu Glu
Phe Ala Lys Gln 865 870 875 880 Pro Lys Thr Ala Asp Tyr Asp Asp Leu
Asp Leu Glu Ile Glu Ala Met 885 890 895 Asn Leu Asp Asp Ile Asp Pro
Ser Asp Leu Asn Leu Asp Glu Asp Leu 900 905 910 Leu Asn Asp 915
95963PRTEuschistus heros 95Met Ala Val Val Glu Gln Pro Cys Tyr Thr
Leu Ile Asn Phe Pro Ser 1 5 10 15 Asp Leu Glu Pro Pro Asn Glu Met
Gln Leu Lys Ser Asp Leu Glu Asn 20 25 30 Gly Asp Thr Lys Thr Lys
Ile Glu Ala Leu Lys Asn Ile Ile His Leu 35 40 45 Ile Ala Asn Gly
Glu Arg Leu Pro Gly Leu Leu Met His Ile Ile Arg 50 55 60 Phe Val
Leu Pro Ser Gln Asp His Thr Ile Lys Lys Leu Leu Leu Ile 65 70 75 80
Phe Trp Glu Ile Val Pro Lys Thr Thr Pro Asp Gly Lys Leu Leu Gln 85
90 95 Glu Met Ile Leu Val Cys Asp Ala Tyr Arg Lys Asp Leu Gln His
Pro 100 105 110 Asn Glu Phe Val Arg Gly Ser Thr Leu Arg Phe Leu Cys
Lys Leu Lys 115 120 125 Glu Pro Glu Leu Leu Glu Pro Leu Met Pro Ala
Ile Arg Ala Cys Leu 130 135 140 Glu His Arg Val Ser Tyr Val Arg Arg
Asn Ala Val Leu Ala Ile Phe 145 150 155 160 Thr Ile Tyr Arg Asn Phe
Glu Phe Leu Ala Pro Asp Ala Pro Glu Leu 165 170 175 Ile Ala Asn Phe
Leu Asp Gly Glu Gln Asp Met Ser Cys Lys Arg Asn 180 185 190 Ala Phe
Leu Met Leu Leu His Ala Asp Gln Glu Arg Ala Leu Ser Tyr 195 200 205
Leu Ala Ser Cys Leu Asp Gln Val Thr Ser Phe Gly Asp Ile Leu Gln 210
215 220 Leu Val Ile Val Glu Leu Ile Tyr Lys Val Cys His Ala Asn Pro
Ser 225 230 235 240 Glu Arg Ser Arg Phe Ile Arg Cys Ile Tyr Asn Leu
Leu Asn Ser Asn 245 250 255 Ser Pro Ala Val Arg Tyr Glu Ala Ala Gly
Thr Leu Ile Thr Leu Ser 260 265 270 Asn Ala Pro Thr Ala Ile Lys Ala
Ala Ala Ser Cys Tyr Ile Asp Leu 275 280 285 Ile Ile Lys Glu Ser Asp
Asn Asn Val Lys Leu Ile Val Leu Asp Arg 290 295 300 Leu Ile Ser Leu
Lys Glu Ile Pro Thr His Glu Arg Val Leu Gln Asp 305 310 315 320 Leu
Val Met Asp Ile Leu Arg Val Leu Ala Ser Pro Asp Met Glu Val 325 330
335 Lys Lys Lys Val Leu Ser Leu Ala Leu Asp Leu Thr Thr Ser Arg Cys
340 345 350 Val Glu Glu Met Val Leu Met Leu Lys Lys Glu Val Ala Lys
Thr His 355 360 365 Asn Leu Thr Glu His Glu Asp Ala Gly Lys Tyr Arg
Gln Leu Leu Val 370 375 380 Arg Thr Leu His Ser Cys Cys Met Lys Phe
Pro Asp Val Ala Ala Ser 385 390 395 400 Val Ile Pro Val Leu Met Glu
Phe Leu Ser Asp Thr Ser Glu Leu Ala 405 410 415 Ser Tyr Asp Val Leu
Ile Phe Val Arg Glu Ala Ile His Lys Phe Asp 420 425 430 Ser Leu Arg
Thr Leu Ile Ile Glu Lys Leu Leu Glu Ala Phe Pro Thr 435 440 445 Ile
Lys Ser Met Lys Val His Arg Ala Ala Leu Trp Ile Leu Gly Glu 450 455
460 Tyr Thr Thr Ser Val Gly Asp Ile Lys Glu Val Met Lys Gln Ile Lys
465 470 475 480 Asn Ala Leu Gly Glu Met Pro Leu Val Asp Asp Glu Met
Lys Arg Ala 485 490 495 Ser Gly Gly Glu Lys Val Glu Glu Val Asp His
Arg Asp Gln Met Lys 500 505 510 Leu Val Thr Ser Asp Gly Thr Tyr Ala
Thr Gln Ser Ile Phe Asn Thr 515 520 525 Ile Leu Ala Ile Lys Lys Glu
Asp Arg Pro Pro Leu Arg Gln Tyr Leu 530 535 540 Ile Asp Gly Asp Phe
Phe Ile Gly Val Ser Val Ala Ser Thr Leu Val 545 550 555 560 Lys Leu
Ala Leu Arg Phe Lys Glu Leu Val Gln Gln Glu Asn Met Tyr 565 570 575
Asn Arg Phe Phe Ala Glu Cys Met Leu Ile Ile Ser Ser Ile Val Arg 580
585 590 Leu Gly Lys Ser Asp Lys Ser Leu Phe Tyr Leu Gly Tyr Pro Ser
Lys 595 600 605 Gln Leu Ser Tyr Asp Asp Tyr Glu Arg Met Leu Leu Cys
Leu Lys Val 610 615 620 Leu Ser Glu Asn Ser Ala Pro Ile Val Lys Ile
Phe Asn Thr Asp Cys 625 630 635 640 Arg Asn Ala Leu Ala Asn Met Leu
Val Ala Gln Gln Asn Glu Glu Tyr 645 650 655 Ser Leu Ile Lys Ala Lys
Glu Lys Ser Val His Thr Ile Gln Val Asp 660 665 670 Asp Pro Val Ser
Phe Leu Gln Leu Ser Thr Met Arg Ser Ser Asp Tyr 675 680 685 Gly Ser
Glu Asn Val Phe Glu Leu Ser Leu Asn Gln Ala Val Gly Gly 690 695 700
Pro Asn Thr Ala Thr Asn Thr Ser Glu Leu Pro Phe Ser Ala Ser Lys 705
710 715 720 Leu Asn Lys Val Thr Gln Leu Thr Gly Phe Ser Asp Pro Val
Tyr Ala 725 730 735 Glu Ala Tyr Val His Val Asn Gln Tyr Asp Ile Val
Leu Asp Val Leu 740 745 750 Ile Val Asn Gln Thr Gly Asp Thr Leu Gln
Asn Cys Thr Leu Glu Leu 755 760 765 Ala Thr Leu Gly Asp Leu Lys Leu
Val Glu Lys Pro Gln Pro Cys Val 770 775 780 Leu Ala Pro Tyr Asp Phe
Cys Asn Ile Lys Ala Asn Val Lys Val Ala 785 790 795 800 Ser Thr Glu
Asn Gly Ile Ile Phe Gly Asn Ile Val Tyr Asp Ile Ser 805 810 815 Gly
Ala Ala Ser Asp Arg Asn Val Val Val Leu Asn Asp Ile His Ile 820 825
830 Asp Ile Met Asp Tyr Ile Val Pro Ala Thr Cys Ser Asp Thr Glu Phe
835 840 845 Arg Gln Met Trp Ala Glu Phe Glu Trp Glu Asn Lys Val Ser
Val Asn 850 855 860 Thr Tyr Leu Val Asp Leu His Glu Tyr Leu Gly His
Leu Leu Lys Ser 865 870 875 880 Thr Asn Met Lys Cys Leu Thr Pro Glu
Lys Ala Leu Cys Gly Gln Cys 885 890 895 Gly Phe Met Ala Ala Asn Met
Tyr Ala Arg Ser Ile Phe Gly Glu Asp 900 905 910 Ala Leu Ala Asn Leu
Ser Ile Glu Lys Pro Phe Asn Lys Pro Asn Ala 915 920 925 Pro Val Thr
Gly His Ile Arg Ile Arg Ala Lys Ser Gln Gly Met Ala 930 935 940 Leu
Ser Leu Gly Asp Lys Ile Asn Met Thr Gln Lys Lys Pro Val Ile 945 950
955 960 Met Ala Gln 96410DNAArtificial Sequencesynthesized
artificial sequence 96atgtcatctg gagcacttct ctttcatggg aagattcctt
acgttgtgga gatggaaggg 60aatgttgatg gccacacctt tagcatacgt gggaaaggct
acggagatgc ctcagtggga 120aagtccggca acatgtttga cgtttgtttg
acgttgtaag tctgattttt gactcttctt 180ttttctccgt cacaatttct
acttccaact aaaatgctaa gaacatggtt ataacttttt 240ttttataact
taatatgtga tttggaccca gcagatagag ctcattactt tcccactgag
300gcatctccgt agcctttccc acgtatgcta aaggtgtggc catcaacatt
cccttccatc 360tccacaacgt aaggaatctt cccatgaaag agaagtgctc
cagatgacat 410
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